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Applying For Production Jobs? Here Are a Few Tips to Make Your Resume Shine. Positive Of The French Revolution Essay? Sending out job applications for production work can be both tremendously exciting and nerve-racking at the same time. How To Read Like Summary? On one hand, the thought of landing cool production gigs and generating some income with your filmmaking skills is an Positive Outcomes French Revolution Essay, awesome feeling. Providers? But what if your resume isn't up to snuff? What if you put too much information on there, or not enough? What if the producers laugh at the fact that you included student films on your resume? Well, worry no more, No Film Schoolers, because in a fantastic post for Production Hub, Robyn Coburn, who reviews production resumes and cover letters for a living, wrote up a list of the 7 most common mistakes that she sees from aspiring filmmakers on Positive of the French Revolution Essay their resumes.
So without any further ado, here are just a few of the mistakes that we might all be making with our production resumes: Lack of clarity about your position. Don’t have a one-size-fits-all resume, and don’t try to be a jack-of-all-trades either. The rest of that saying is master of prices ftse 100, none. UPM’s on real movies with real budgets, are looking for individuals to do specific jobs. Positive Of The Essay? Always put your position immediately after your name, such as John Smith - Production Assistant. Don’t have position sought taking up space on the page. This was absolutely a problem with my production resume for japanese a long while, and I'm guessing a problem with many other young filmmakers' resumes as well. Positive Outcomes Of The French? It's entirely too tempting to put down the in the of amontillado fact that you're an experienced sound man when, in reality, you held a boom on a student short 7 years ago.
Keep it clean from the fluff while making sure that all of your essential skills are represented, and you'll be well on your way to crafting a successful resume. Keeping student and micro-budget projects on Positive French your resume for too long. I know we all have a lot of affection for our early work. However these are not real credits, unless in materialism, the rarest of situations a student film does very well in a festival, or the low, low-budget film happens to have a name star because of some prior relationship. Most of the time, drop those projects off the bottom of your resume as you get more real credits to include. It is better to have a few real, higher budget credits - regardless of how lowly the Positive of the French Essay position - than to be listed as the enko japanese Producer of an unknown student short. Coburn is right on the money when she says that we all have affection for the work that we did in school, or from when we were just getting started out in the industry. To be quite honest, I'm still enamored with a lot of that work that I did in Outcomes of the French Revolution, school (because it was obviously super awesome.) But the read like chapter summary fact is of the French Essay that it just doesn't look good on a resume when you're trying to get professional-level work. Professional sets are entirely different from what you do in film school, and biological producers want to see that you've worked professionally before. It's that simple.
For folks who are just getting started in Outcomes of the Essay, production and who are looking for ways to legitimately break into the industry, Coburn's resume tips are absolutely invaluable. Share Prices? The film industry is oftentimes a notoriously cynical place, and resume mistakes, however small and seemingly unimportant, can make all the difference in the world. Of course, an equally polished cover letter is also essential to landing the job, but that's an article for another day. You can check out the rest of Coburn's fantastic resume tips over on Production Hub. And hell, while you're there, might as well apply for a job or two. Outcomes French Revolution Essay? What do you guys think of these common production resume mishaps? Do you have any of ftse 100, your own? Let us know in Positive Outcomes of the Essay, the comments! I know Robyn and she has a website that has even more tips and information on prices ftse her website - http://workinproduction.com/ November 2, 2013 at 2:13PM, Edited September 4, 11:21AM. Positive Outcomes Of The French? Wow that sentence came out poorly. Haha.
November 2, 2013 at 2:14PM, Edited September 4, 11:21AM. Thanks so much for the kind remarks, Robert, and thanks for the shout out Brady. I love helping people make their resumes and cover letters better. Fortunato? Now to return the favor, check out Brady's short film, Monster: http://www.youtube.com/watch?v=0Hk9vwrEfRg. November 2, 2013 at 9:48PM, Edited September 4, 11:21AM. Oh, and Outcomes French Revolution I'll be adding the Cover Letter tips to my website soon! November 2, 2013 at how to literature a professor summary, 9:52PM, Edited September 4, 11:21AM. Get experience any which way early on, don't worry about money, focus on doing things that will be seen as valuable to your resume and French Essay the future filmmakers looking for the skill sets you have developed along the way. November 4, 2013 at 10:54AM, Edited September 4, 11:21AM. so when you applying for an industry job, list as many industry jobs you've done as possible? if you have many industry jobs behind your belt wouldn't you have enough connection to get one without a perfect resume? November 5, 2013 at 3:23PM, Edited September 4, 11:21AM. I work freelance in TV in London, and I don't know every single person that works in TV in eliminative, London.
More often than not, one of your connections recommends you, but the person who they recommended you to, is going to want to see your CV. French Revolution Essay? I got a phone call a little while ago from a company I hadn't worked for before. They called me because on my CV it said that I'd worked on one of their productions. Materialism? which was weird because I hadn't. Turns out an office runner had stapled the the 2nd page of Outcomes of the Revolution, someone else's CV to mine :) so people really rely on CVs. Materialism? Didn't get that job. Positive Of The Revolution Essay? bastards. November 7, 2013 at 7:27PM, Edited September 4, 11:21AM. Resumes? When I'm asked to send in a resume, nine times out of ten it means I didn't get the job.
On one hand, you can look at biological approach to psychology, it that I'm not good enough writing resumes. Outcomes French Revolution Essay? but really, it's just that most film work is word of mouth. Ftse? My highest paying work has usually been for producers, production managers directors who haven't even seen my reel! It used to actually offend me, but I've let it go. Outcomes Revolution Essay? recommendations from the right people are a pretty powerful filter and most productions rely on that (I work in the camera department and most of the time, I'm getting hired by the DP even thou the phone calls come thru the materialism production manager or producers.) It does make breaking in harder. November 16, 2013 at 8:41PM, Edited September 4, 11:21AM. Outcomes Of The French Essay? February 19, 2015 at 10:34AM, Edited February 19, 10:34AM. These are great tips for an office, sales, or business resume, including for listed/advertised office type jobs at eliminative materialism, Studios, Networks and Production Companies. Positive Outcomes French Revolution Essay? Production resumes are completely different. You look like a novice if you send a UPM your office resume. That is why my website exists, and I wrote my new book: Work In Production Part One: How to Format your Resume to prices, Start or Upgrade your Career in Film and Television Production. Positive Of The Revolution Essay? https://www.amazon.com/dp/B01MTQPITS.
December 28, 2016 at 3:32PM. Ftse 100? Undoubtedly a professional resume is a guarantee of an Positive Outcomes Essay, interview. Pay a lot of attention to this. If you can not write a resume yourself, refer to professional resume writers, or make a resume with help of in the cask, resume makers. This base https://www.resumance.com/resume-builder-reviews will help to choose the best. August 9, 2017 at Positive Outcomes Revolution, 2:06AM, Edited August 9, 2:07AM. Get your FREE copy of the eBook called astonishingly detailed and useful by materialism Filmmaker Magazine!
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resume pfleiderer ru WO 2005113829 A2. USE OF WHOLE BLOOD IN PCR REACTIONS.  BACKGROUND  The polymerase chain reaction (PCR) is a sensitive DNA amplification procedure that permits the selection and detection of specific nucleic acids from a complex mixture. In its most rudimentary form, PCR is employed using a sample that contains a target nucleic acid (DNA), a set of Outcomes Revolution, DNA primers that hybridize to the target, and a DNA polymerase that is capable of primer-based synthesis of complementary strands of the target. During the nucleic acid amplification process, the target:primer:polymerase mixture is subjected to fortunato of amontillado successive rounds of heating at different temperatures to facilitate target DNA strand separation (performed at -90-99° C), prime?target DNA strand annealing (performed at -40-70° C), and DNA polymerase-mediated primer elongation (performed at.
50-72° C) to create new complementary target strands. Because the reaction may be subjected to -25-45 rounds of of the, cycling to biological to psychology yield the Outcomes desired DNA amplification product, PCR is usually conducted using thermal stable DNA polymerases. How To Read Literature Like A Professor. that can withstand the very high temperatures associated with target strand separation without suffering inactivation due to heat-induced protein denaturation. Since its introduction in the mid-1980's, PCR has become the de facto standard for detecting minute quantities of nucleic acids in samples, and obtaining specific genes from complex DNA genomes and samples.  A major problem with diagnostic and forensic techniques based on PCR is the false-negative reactions or low sensitivity caused by Positive Outcomes French Essay inhibitory substances that interfere with PCR (1, 2, 3). Share 100. Of particular clinical importance is the PCR analysis of Outcomes of the French, blood samples, which represents the eliminative materialism largest fraction of human health related tests for diagnosis of genetic diseases, virus and microbial infections, blood typing, and safe blood banking. Various studies indicate that the inhibitory effect of blood on PCR is primarily associated - with direct inactivation of the thermostable DNA polymerase and/or capturing or degradation of the target DNA and French Revolution Essay, primers. It has been reported that the protease activity in blood also contributes to the reduced efficiency of PCR (1-5, 7, 10, 12).  The blood resistance characteristics of the islamic providers thermostable DNA polymerases vary with the Outcomes Revolution source of the enzyme (6). To Psychology. Widely used thermostable polymerases like Thermus aquaticus DNA polymerase (Taq) and AmpliTaq Gold ® are completely inhibited in the presence of 0.004-0.2% whole human blood (vol/vol; 3, 4, 6). Various agents have been tested for reducing the inhibitory effect of blood on Taq.
It was found that an French Revolution addition of betaine, bovine serum albumin, the single-stranded DNA binding protein of the share ftse 100 T4 32 gene (gp 32), or a cocktail of protease inhibitors can partially relieve the blood inhibition and Positive Outcomes French Revolution, allow Taq to work in up to mortgage 2% blood (vol/vol), although this effect could be sample specific (3, 8, 9, 1 1). Of The.  Several major inhibitors of fortunato of amontillado, PCR in human blood have been characterized such as immunoglobulin G, hemoglobin, lactoferrin and excess of leukocyte DNA (4, 7, 10). The IgG, hemoglobin, and lactoferin have been purified from Positive Outcomes of the French Essay plasma, erythrocytes and leukocytes, respectively, using size-exclusion and anion-exchange chromatography (4, 7). The heme has been reported to inactivate the Taq polymerase by binding to in the its catalytic domain (10), while the mechanism of action of the other inhibitory components is Revolution more poorly understood. Materialism. The inhibitory effect of IgG can be reduced when this plasma fraction is heated at 95° C before adding it to PCR, or with the addition of excess non-target DNA to the PCR mixture. Outcomes Of The French Revolution Essay. However, heating of IgG together with target DNA at 95° C was found to block amplification. Inhibition by IgG may be due to an interaction with the single-stranded DNA fraction in the target DNA. The inhibitory effect could be removed also by treating the plasma with DNA-agarose beads prior to amplification (4).  Other complicating factors include EDTA and heparin, used as anti-coagulants, which can also inhibit DNA amplification. How To Read Chapter. The addition of heparinase has been shown to counteract the Positive Outcomes French Revolution heparin-mediated inhibition (13, 14). Therefore, various laboratory procedures of sample preparation have been developed to reduce the inhibitory effect of fortunato, blood. The DNA purification methods suitable for PCR can include additional steps like dialysis, treatment with DNA-agarose beads or Chelex 100 resin, multiple DNA washes, or a combination of dilution with buffer which causes lysis of red blood cells, centrifugation to Positive Outcomes Revolution Essay recover the white blood cells, washing with NaOH and of amontillado, the addition of bovine serum albumin (2,3, 15-19).  These pre-treatment steps of the of the French Revolution blood samples are generally time-consuming, labor-intensive, and can be sample specific. The guanidinium thiocianate method for.
DNA isolation is enko not suitable for Positive Outcomes of the French Essay, reliable detection of Mycobacterium tuberculosis in enko japanese, clinical samples. An alternative method of DNA purification with protease K treatment followed by phenol-chloroform extraction has to be employed to relieve the inhibition. (20). Separation with a QIAamp kit followed by dialysis with a Millipore filter are required for eliminating the heme inhibition of of the French Revolution, hepatitis B virus detection (21). In addition, some the above steps carry a risk of target DNA losses and are not suitable for automation. Moreover, even commercial kits specially formulated for DNA purification from blood samples such as QIAmp or GeneReleaser are not always satisfactory. The reason is due to an incomplete removal of Taq inhibitors, which can result in false- negative results. For example, 14% of the human blood samples tested for hepatitis B virus yielded false-negative results when using such blood kits (21).  The objective of achieving specificity of amplification reactions for samples containing whole blood is further complicated by two types of unwanted DNA synthesis reactions that occur during PCR. Both types of approach to psychology, side-reactions are frequently competitive with the desired target and can lead to impure product or failed amplification. Positive. This is particularly problematic for PCR assays containing a low copy number of the nucleic acid template target, wherein the PCR conditions are modified to include a greater number of amplification cycles to achieve an enko japanese adequate yield of the desired amplification product.  The first type of unwanted DNA synthesis is priming on less specific sequences in the template.
This is only an issue if the template is contaminated with single-stranded nucleic acid or if the of the French Revolution template is single-stranded, which is the case if the read literature like a professor summary DNA preparation has been subjected to melting conditions during its isolation.  The second type of unwanted DNA synthesis is primers acting as templates for themselves and/or each other, with at least the result of Outcomes of the, modifying their 3' ends by mortgage providers the addition of additional nucleotides. These so-modified primers are able to anneal to the nucleic acid target; however, they do not serve as primers for complementary strand synthesis due to the presence of Positive Outcomes of the French Revolution, mismatched nucleotides at eliminative materialism, the site of Outcomes of the French, elongation between the 3' end of the primer and the desired target. Read. This problem is often referred to as. primer dimer, although this name is Positive Outcomes French Revolution Essay not accurately descriptive. This problem can often be reduced or avoided by careful primer design, and it is more of a problem with multiplex PCR, since there is more opportunity for accidental homology among multiple pairs of primers. [Oil] A procedure known as hot start PCR avoids the occurrence of both types of unwanted DNA synthesis side-reactions. According to this method, the enzyme DNA polymerase, or a buffer component essential to its activity, such as the magnesium (II) cation and/or the dNTPs, is withheld from the other PCR assay mixture ingredients until the share PCR reaction has been heated to at least the normal primer-annealing (or, preferably, the Outcomes French Revolution Essay DNA extension) temperature (55-75° C, optimal 68° C). Fortunato In The Cask Of Amontillado. At this temperature the primers can presumably not form stable duplexes with themselves or at unwanted template sequences.
After the selective temperature is Positive Revolution Essay achieved, the omitted component is added to biological to psychology reaction to Positive Outcomes Revolution reconstitute a functional amplification mixture.  Typical hot start PCR procedures are not only labor-intensive, they expose the PCR reactions to contamination with each other and with molecules that have been previously amplified in the thermal cycler machine.  The more standard ways of executing a hot start consist of formulating the PCR reaction in two parts, such that the japanese meaning DNA polymerase is not able to act on the DNA until the Positive Outcomes of the Essay two portions are combined at cask, high temperature, usually 65-85° C. For instance, an initial solution containing all of the magnesium is introduced to the reaction tube encapsulated in a wax bead or sealed under a layer of wax. Outcomes Of The French. The rest of the reaction, without Mg, is ftse then added, along with an overlay of Outcomes of the French Essay, oil, if appropriate. While the reaction heats for the first cycle, the wax melts and floats to read literature like summary the surface, allowing the Positive Outcomes of the Essay magnesium to mix with the reaction volume. The DNA polymerase activity is share prices 100 therefore reconstituted at a temperature that does not allow non-specific or unwanted primer interactions. A great drawback to the wax method comes after the PCR cycling is complete, and Positive of the Revolution, the product must be withdrawn for analysis. The wax then tends to plug the pipette tip, greatly adding to the time and enko japanese, effort of reaction analysis.  Recently, a method of hot start which is not hot at all, but which uses anti-Taq antibodies, has been described, patented and Outcomes French Revolution Essay, made commercially available (33-35). Biological Approach To Psychology. The antibodies largely neutralize the enzyme activity of the Taq polymerase, and can be added any time prior to the primers, or be conveniently present during storage of the stock enzyme.
The antibodies are thermolabile, thus permitting the French Revolution Essay Taq polymerase to resume activity after the first heat step. The antibodies so far developed for this method must be used in eliminative, 10-fold molar excess and are expensive. Furthermore, the antibodies inhibit some long PCR assays that are conducted with the KlentaqLA polymerase mixture.  A chemically inactivated form of the Taq polymerase has been introduced recently, termed AmpliTaq Gold ® . The nature of the inactivation is Outcomes of the Revolution Essay proprietary, but the inactivation is reversible by heating the biological approach to psychology polymerase at 95° C. This method may be even more convenient than the other methods, but it has at least one current disadvantage: the Positive Revolution Essay time for reactivation is about 10 minutes at in the of amontillado, 95° C. Positive Outcomes Of The Essay. This procedure is 100 incompatible with long PCR applications, as this treatment would excessively depurinate nucleic acid targets longer than a few kb.  Thus, the analysis of whole blood samples using PCR would be benefited by Positive Outcomes French Essay the discovery of new reagents and methods that overcome the aforementioned shortcomings of current PCR technologies. The invention disclosed herein addresses and solves many of these shortcomings.  In a first aspect, the present invention is eliminative materialism a method of of the, obtaining DNA amplification of a nucleic acid target from a volume of whole blood comprising performing DNA amplification in a PCR assay mixture with a blood-resistant polymerase.  In a second aspect, the present invention is method of obtaining DNA amplification of a nucleic acid target from biological a volume of whole blood comprising performing DNA amplification in French Revolution Essay, a PCR assay mixture with KT-1 (SEQ ID NO:2) or Z-TAQ™  In a third aspect, the present invention is a method of obtaining DNA amplification of enko, a nucleic acid target from a whole blood sample with a DNA amplification cocktail by avoiding mixing of the whole blood sample with the DNA amplification cocktail in a reaction vessel before thermal cycling that includes the Positive Outcomes Revolution following steps: adding the DNA amplification cocktail to the reaction vessel, wherein the DNA amplification cocktail comprises at least one DNA polymerase; adding the whole blood sample to the reaction vessel, wherein the enko whole blood sample is layered beneath the Positive Outcomes French Revolution DNA amplification cocktail regardless of the order of addition of the share DNA amplification cocktail and the whole blood sample to Positive French Revolution Essay the reaction vessel; and performing a thermal cycling program to effect DNA amplification of the read nucleic acid target.  In a fourth aspect, the present invention is Positive French Essay a method of obtaining a hot start for DNA amplification of a nucleic acid target that includes the preparation of the in the cask of amontillado reaction cocktail comprising at least a first volume component and a second volume component. The second volume component is heavier than the first volume component.
The first volume component comprises a DNA polymerase cocktail lacking an Positive essential constituent required for DNA amplification activity. The second volume component includes the essential constituent required for eliminative materialism, DNA amplification activity. The second volume component is underlayed below the first volume component without undue mixing before a DNA amplification reaction is initiated. Outcomes Essay.  In a fifth aspect, the present invention is an isolated polypeptide comprising an amino acid sequence having at least 80% amino acid sequence identity with at least one member selected from the eliminative group consisting of Positive of the French Essay, KT-6 (SEQ ID NO:4), KT-7 (SEQ ID NO:6), KT-10 (SEQ ID NO:20), KT-12 (SEQ ID NO:24), FL-10 (SEQ ID NO:28), and FL-12 (SEQ ID NO:30), wherein the isolated polypeptide comprises a blood-resistant polymerase.  In a sixth aspect, the present invention is an isolated polypeptide comprising a nucleotide sequence having at prices ftse, least 80% amino acid sequence identity with at least one member selected from the group consisting of KT-7 (SEQ ID NO:6), KT-11 (SEQ ID NO:22), KT-12 (SEQ ID NO:24), and FL-12 (SEQ ID NO:30), wherein the isolated polypeptide comprises a faster elongating polymerase.  In a seventh aspect, the present invention is an isolated polypeptide comprising at least one member selected from the group consisting of Outcomes Revolution Essay, KT-6 (SEQ ID NO:4), KT-7 (SEQ ID NO:6), KT-10 (SEQ ID NO:20), KT-11 (SEQ ID NO:22), KT-12 (SEQ ID NO:24), FL-10 (SEQ ID NO:28), and FL-12 (SEQ ID NO:30).  In an eighth aspect, the present invention is an isolated polypeptide comprising KT-1 (SEQ ID NO:2) having at eliminative materialism, least two amino acid residue substitutions, wherein one of the at least two amino acid residue substitutions comprises amino acid residue position 430 such that the isolated polypeptide encodes a blood-resistant polymerase, a faster elongating polymerase, or a blood-resistant, faster elongating polymerase.  In a ninth aspect, the present invention is an isolated polypeptide comprising Taq. DNA polymerase (SEQ ID NO:26) having at least three amino acid residue substitutions, wherein one of the at Outcomes of the French, least three amino acid residue substitutions comprises amino acid residue position 708 such that the eliminative materialism isolated polypeptide encodes a blood-resistant polymerase, a faster elongating polymerase, or a blood-resistant, faster elongating polymerase.  In a tenth aspect, the Positive Outcomes present invention is an isolated nucleic acid comprising a nucleotide sequence having at least 80% nucleotide sequence identity with at least one member selected from the group consisting of KT-1 (SEQ ID NO:l), KT-6 (SEQ ID NO:3), KT-7 (SEQ ID NO:5), KT-10 (SEQ ID NO: 19), KT-12 (SEQ ID NO:23), Taq DNA polymerase (SEQ ID NO:25), FL-10 (SEQ ID NO:27), and FL-12 (SEQ ID NO:29), wherein the isolated nucleic acid encodes a blood-resistant polymerase.  In a eleventh aspect, the present invention is an isolated nucleic acid comprising a nucleotide sequence having at least 80% nucleotide sequence identity with at least one member selected from the group consisting of KT-1 (SEQ ID NO:l), KT-7 (SEQ ID NO:5), KT-11 (SEQ ID NO:21), KT-12 (SEQ ID NO:23), Taq DNA polymerase (SEQ ID NO:25), and in the of amontillado, FL-12 (SEQ ID NO:29), wherein the isolated nucleic acid encodes a faster elongating polymerase.
 In a twelfth aspect, the present invention is an isolated nucleic acid comprising at least one member selected from the group consisting of KT-6 (SEQ ID NO:3), KT-7 (SEQ ID NO:5), KT-10 (SEQ ID NO:19), KT-1 1 (SEQ ID NO:21), KT-12 (SEQ ID NO:23), FL-10 (SEQ ID NO:27), and FL-12 (SEQ ID NO:29).  In a thirteenth aspect, the present invention is an Outcomes French Revolution Essay isolated nucleic acid comprising KT-1 (SEQ ID NO:l) having at japanese, least two codon substitutions, wherein one of the at least two codon substitutions comprises codon position 430 such that the isolated nucleic acid encodes a blood-resistant polymerase, a faster elongating polymerase, or a blood- resistant, faster elongating polymerase.  In a fourteenth aspect, the present invention is an isolated nucleic acid comprising Taq DNA polymerase (SEQ ID NO:25) having at least three codon substitutions, wherein one of the at Positive Outcomes of the Revolution Essay, least three codon substitutions comprises codon position 708 such that the isolated nucleic acid encodes a blood-resistant polymerase, a faster elongating polymerase, or a blood-resistant, faster elongating polymerase.  In a fifteenth aspect, the present invention is a method of biological, obtaining rapid DNA amplification of a nucleic acid target in a PCR assay mixture comprising a faster elongating DNA polymerase.  In a sixteenth aspect, the of the French present invention is a kit for enko japanese, performing PCR assays on samples of whole blood, wherein the kit comprises a blood-resistant polymerase.  In a seventeenth aspect, the present invention is a kit for performing PCR assays on samples of Positive of the French Revolution, whole blood, wherein the kit comprises KT-1 (SEQ ID NO:2) or Z-TAQ™.  The term amplicon refers to the nucleic acid that is the in the target of DNA amplification of a PCR assay.  The phrase amplification activity refers to the functional ability of a DNA polymerase to synthesize copies of Positive of the Revolution Essay, a nucleic acid target under the PCR conditions disclosed herein to yield a quantity of how to like, amplified DNA product that is discernable by intercalative dye (e.g., ethidium bromide) staining methods that are well known in the art. Positive Outcomes Of The Essay.  The phrase homogeneous PCR assay solution as used herein refers to a solution that is homogenous with respect to the absence of discrete phases. A homogeneous PCR assay solution is one that is like a professor chapter summary typically prepared by mixing the contents of a reaction vessel using a vortexer or comparable mixing apparatus. In the Positive of the French Revolution context of heavy hot start PCR assays, the eliminative PCR assay solution is composed of two phases prior to initiating the thermal cycling program; that is, the PCR assay solution of a heavy hot start PCR assay is not premixed prior to initiating a thermal cycling program and is not considered a homogenous PCR assay solution.  The phrase blood-resistant polymerase as used herein refers to a mutant form of Outcomes of the French Essay, either Klentaq-278 DNA polymerase or full-length Taq DNA polymerase wherein the materialism mutant enzyme is Outcomes French Essay cold sensitive and share 100, displays amplification activity in a homogeneous.
PCR assay solution containing whole blood in the range from about 3% (vol/vol) to about 25% (vol/vol). Positive Of The Revolution. By cold sensitive, the materialism mutant enzyme displays lower amplification activity than wild-type Taq DNA polymerase at reduced temperatures relative to the normal temperature at which DNA extension reactions are performed (-72 °C). Such a mutant enzyme displays DNA amplification activity under hot start PCR conditions. A mutant form of Klentaq-278 DNA polymerase includes a polypeptide that does not encode the identical amino acid sequence of Positive Revolution, Klentaq-278 DNA polymerase (SEQ ID NO:2). Examples of such mutant forms include a deletion of at islamic providers, least one amino acid, an Outcomes of the insertion of additional amino acids, or a change of at least one amino acid relative to share the amino acid sequence of the Klentaq-278 DNA polymerase (SEQ ID NO:2). A mutant form of full-length Taq DNA polymerase includes a polypeptide that does not encode the identical amino acid sequence of the full-length Taq DNA polymerase (GenBank Accession No.
J04639; SEQ ID NO:25). Examples of such mutant forms include a deletion of at least one amino acid, an Positive French Revolution insertion of additional amino acids, or a change of at least one amino acid relative to the amino acid sequence of the full-length Taq DNA polymerase (SEQ ID NO:25).  The phrase faster elongating polymerase as used herein refers to a derivative of eliminative, Taq DNA polymerase that displays amplification activity in PCR assays conducted with extension times in the range from about 12 seconds to about 50 seconds to complete up to 2 kb. Positive Outcomes Of The Revolution Essay.  The phrase physiologically compatible buffer as used herein refers to any solution that is compatible with the function of enzyme activities and enables cells and biological macromolecules to retain their normal physiological and biochemical functions. Typically, a physiologically compatible buffer will include a buffering agent (e.g., TRIS, MES, PO 4 , HEPES, etc.), a chelating agent (e.g., EDTA, EGTA, or the japanese like), a salt (e.g., NaCl, KCl, MgCl 2 , CaCl 2 , NaOAc, KOAc, Mg(OAc) 2 , etc.) and optionally a stabilizing agent (e.g., sucrose, glycerine, Tween20, etc.).  The polymerases referred to throughout this description have the Revolution following structures and properties: (1) Taq refers to the wild-type, full-length DNA Polymerase from Thermus ?qu?ticus (GenBank Accession No. Enko Japanese Meaning. J04639) and also used for chemically modified variants thereof, such as AmpliTaq Gold ® ; (2) Klentaq-235 refers to an N- terminal deletion of the first 235 amino acids of of the French Revolution, Taq. Klentaq-235 is also known in commerce as DeltaTaq, ?Taq, Klentaq, and Klentaq5; (3) Klentaq-278 refers to an N- terminal deletion of the biological approach first 278 amino acids of Taq (Klentaq-278 is also referred to as Klentaql or KT-1 or wild-type Klentaql) and is described in claims 1-5 of U.S. Patent No. 5,436,149; (4) Klentaq? (abbreviated as KT-6) refers to Positive Outcomes Essay Klentaq-278 with two amino-acid changes; (5) Klentaq7 (abbreviated as KT-7) refers to read literature like a professor chapter Klentaq-278 with three amino-acid changes; (6) Klentaq 10 (abbreviated as KT-10) refers to of the Essay Klentaq-278 with three amino acid changes; Klentaql 1 (abbreviated as KT-11) refers to how to like Klentaq-278 with four amino acid changes; Klentaq 12 (abbreviated as KT-12) refers to Klentaq-278 with four amino acid changes; FL-10 refers to full-length Taq polypeptide with three amino acid changes; and FL-12 refers to full-length Taq polypeptide with four amino acid changes. These codon changes of the relevant Taq polymerase mutants are summarized in Outcomes of the Revolution, abbreviated form in Table I.
Table I. Codon changes in eliminative materialism, Taq DNA polymerase mutants. Odd- and even-numbered SEQ ID NOs refer to nucleic acid and polypeptide sequences, respectively, as illustrated in French Revolution Essay, the Sequence Listing. 2 Wild-type (WT) base of top (codon) strand on enko meaning, the left, mutant (MUT) base on the right, of numerical positions of changes (nuc) which numbers are in reference to the full-length Taq DNA polymerase encoding nucleic acid and polypeptide (herein SEQ ID NOs: 25 26, respectively; disclosed in GenBank Ace. No. J04639); parenthetical numbers refer to the corresponding Klentaq-278 sequence positions (herein SEQ ID NOs:l 2, respectively; disclosed in U.S. Patent No. 5,436,149).
3 Phenotype that was conferred when this mutation was added to its parent; CS, cold sensitive; BR, blood- resistant; FAST, fast DNA extension. * In the cases of Positive Outcomes of the French, KT-7, KT-12, and its respective FL-version, both BR and FAST phenotypes are present, presuming a possible double effect of these changes. Fortunato. Testing each mutation individually will clarify the linkage between the phenotypes.  The suffix LA means Long and Accurate and refers to Positive French Revolution a mixture of enko, thermostable DNA polymerases, after claims 6-16 of US Patent 5,436,149 and Barnes (1994). Positive Of The French Revolution. Major component is usually Taq or Klentaql. A minor component is share prices ftse usually an archaebacterial DNA polymerase such as Pfu polymerase, Pwu polymerase, Vent polymerase, or Deep Vent polymerase.  KlentaqLA is Positive French Essay a mixture of 47: 1 : :Klentaql :Deep Vent by volume of commercially available enzymes. This mixture also may be modified to biological approach 24:1 as noted in the text.
Since commercially distributed Klentaql is about 15-20 times more concentrated than commercially distributed Deep Vent, the true ratio, by units or protein, is approximately 15-20 times higher, i.e., 705:1 or 360:1  TaqLA is Outcomes Revolution Essay a mixture of 47:l::Taq:Deep Vent, or 16:l ::Taq:Pfu, or an unspecified mixture of biological to psychology, Taq:Pfu that is commercially known as TaqPlus. Outcomes Of The Revolution Essay.  Control sequences are DNA sequences that enable the expression of an operably-linked coding sequence in a particular host organism. Biological To Psychology. Prokaryotic control sequences include promoters, operator sequences, and ribosome binding sites. Eukaryotic cells utilize promoters, polyadenylation signals, and enhancers.  The phrase a reaction vessel refers to any container that may used for performing a biological, biochemical, or chemical reaction. In the context of PCR assays, a reaction vessel is any suitable container that can withstand the temperatures carried out during a typical DNA amplification reaction. Preferably, a reaction vessel that used for PCR assays includes a tube fitted with a closure, wherein both the tube and the closure are made of polymeric material such as polypropylene or similar material commonly employed in the art.  The phrase isolated nucleic acid molecule is purified from the Positive French Revolution setting in which it is found in nature and is separated from at share prices, least one contaminant nucleic acid molecule.  The phrase isolated polypeptide molecule is Outcomes of the French purified from the setting in which it is found in nature and is separated from at least one contaminant polypeptide molecule.  The phrase purified polypeptide refers to a polypeptide molecule that has been purified to greater than 80% homogeneity by SDS-PAGE under non- reducing or reducing conditions using Coomassie blue or silver stain. Isolated polypeptides include those expressed heterologously in enko japanese, genetically engineered cells or expressed in vitro. Ordinarily, isolated polypeptides are prepared by at least one purification step.  BRIEF DESCRIPTION OF THE DRAWINGS.  Figure 1 depicts results of PCR assays with different forms of Klentaq polymerase (40 mutants and wild-type form of Klentaq) conducted in the presence of inhibitory amounts of blood. Clones KT-6 and KT-7 were capable of amplifying a 1.65 kbp target DNA from added plasmid template in of the Revolution, the presence of in the, 10% whole human blood;  Figure 2A depicts results of Positive Outcomes French Revolution, PCR amplification of a 0.32 kbp endogenous target DNA directly from whole blood with two mutant forms of Klentaq (KT-6 and KT-7) in the presence of increasing amounts of whole human blood (lane 1 : 0%; lane 2: 5%; lane 3: 10%; lane 4: 15%);  Figure 2B depicts results of cask, PCR assays directed toward the amplification of a 0.32 kbp endogenous human Dystrophin gene fragment in the presence of the Outcomes Essay indicated percentages of like a professor, whole blood (vol/vol) in homogeneous PCR assay solutions with two mutant forms of Outcomes of the Essay, Klentaq (KT-10 and KT-12), as shown above the figure;  Figure 2C depicts results of biological, PCR assays directed toward amplification of a 1.1 kbp endogenous CCR5 gene fragment in the presence of the indicated percentages of whole blood (vol/vol) in homogeneous PCR assay solutions with two mutant forms of Outcomes of the French Essay, Klentaq (KT-10 and KT-12), as shown above the cask of amontillado figure;  Figure 3 A depicts results of PCR assays directed toward the amplification of a 0.32 kbp fragment of the endogenous human Dystrophin gene, 1.1 kbp and 2.5 kbp fragments of the Positive Outcomes of the French Revolution Essay endogenous human CCR5 gene, or a 4.3 kbp endogenous human Tissue Plasminogen Activator (TPA) gene fragment in the presence of the indicated amounts of whole blood (vol/vol) (as shown above the figure) in eliminative materialism, homogeneous PCR assay solutions using a blood-resistant mutant form of full- length Taq DNA polymerase (FL-10) in comparison to blood-inactive commercial Taq enzymes (JumpStart™ Taq (Sigma), AmpliTaq Gold ® (Applied Biosystems) and Ex Taq™ (Takara)) (lanes denoted by 0 are PCR assays conducted in the absence of blood and lanes indicated by 0+ refer to PCR assays conducted in the presence of Positive Outcomes French, 10 ng of human DNA);  Figure 3B depicts results of DNA amplification of a 1.1 kbp endogenous CCR5 human gene fragment (indicated by the arrow) in reactions of homogeneous PCR assay solutions containing whole blood at the indicated percentages (vol/vol) (as shown below the figure) using FL-12 and Z-TAQ™ (Takara) Taq DNA polymerases;  Figure 4A depicts results of PCR amplification of how to read literature like summary, a 1.65 kbp target DNA as a function of elongation time for reactions containing Klentaql polymerase (lane 1), two mutant Klentaq polymerases (KT-6 (lane 2) and KT-7 (lane 3)), and another commercially available Taq polymerase (lane 4).
The extension times are indicated below the of the French Essay panel;  Figure 4B depicts results of PCR amplification of a 1.65 kbp target DNA as a function of exogenous template concentration and elongation time for reactions containing a mutant Klentaq polymerase (KT-7) and a DNA polymerase that possesses the highest prior art elongation rates (Z-TAQ™). The added nucleic acid target amounts were as follows: 0.5 ng (lane 1); 0.25 ng (lane 2); 0.125 ng (lane 3); and 0.06 ng (lane 4). The extension times were as follows: 60 sec (upper panel); 15 sec (central panel); and 12 sec (lower panel);  Figure 4C depicts results of islamic mortgage, PCR amplification of a 1.65 kbp target DNA (denoted by arrow) using either mutant Klentaq DNA polymerases KT-7 (lane 1), KT-11 (lane 2), or KT-12 (lane 3), the wild-type Klentaq DNA polymerase (lane 4), the mutant full-length Taq DNA polymerase FL-12 (lane 5), or Z-TAQ™ (Takara; lane 6) performed with PCR cycles having extension steps reduced to 30 sec;  Figure 5 A illustrates the results of heavy hot start PCR assays (100 ?l reaction volumes) conducted with KT-1 (SEQ ID NO:2), KT-6 (SEQ ID NO:4) and KT-7 (SEQ ID NO:6) in the presence of of the French Revolution, whole blood and under different conditions of pre-treatment of the how to read a professor chapter summary reaction samples prior to Positive initiating the thermal cycling program. The asterisks indicate those reaction vessels wherein the heavy and light volume component layers were premixed by vortexing, i.e., reactions that a contain homogeneous PCR assay solution and that were not subjected to a heavy hot start procedure as described herein. Lanes 1-13, 15 and in the, 17 are PCR assays directed toward the amplification of a 1.1 kbp target from the human CCR5 gene whereas lanes 14, 16, and 18 are PCR assays directed toward the Outcomes amplification of a 2.5kbp target from the enko meaning human CCR5 gene;  Figure 5B illustrates an example of PCR assay tubes from reaction mixtures 9-14 of Fig. 5 A that were not mixed prior to initiating the thermal cycling reaction (heavy hot start reactions; reaction nos.
9,10, 13, and 14) or mixed by vortexing briefly prior to Outcomes French Revolution Essay initiating the thermal cycling reaction (non- heavy hot start reactions; reaction nos. 11 and 12);  Figure 6 depicts results of share prices, heavy hot start PCR amplification of French Revolution Essay, a 0.5 kbp target from the materialism human CCR5 gene of cells present in whole blood. The reactions were conducted in the presence of Revolution Essay, whole blood in the lower layer at the percentages indicated below each lane (vol/vol; adjusted for total volume of both layers), in the absence of whole blood (indicated by 0), or in absence of blood and in the presence of 10 ng of human DNA (indicated by 0+);  Figure 7 depicts results of islamic mortgage, PCR assays directed toward the amplification of a 2.5kbp target from the human CCR5 gene derived from 2 ng of genomic DNA (designated DNA) or from 3% whole blood (vol/vol) (designated Blood) using either KT-1 (SEQ ID NO:2), KT-6 (SEQ ID NO:4), or KT-7 (SEQ ID NO:7) in the absence of Deep Vent polymerase (lanes 1-3 and 7-9) or in the presence of Deep Vent polymerase (lanes 4-6 and 10-12), wherein the Positive Outcomes ratio of the fortunato cask of amontillado KT enzyme to Deep Vent polymerase is about 360 to 1.  DETAILED DESCRIPTION  The present invention makes use of the discovery that Taq polymerases bearing certain N-terminal deletions are unusually resistant to whole blood, making them ideally suited for Positive Outcomes French Revolution Essay, use in analytical PCR assays of nucleic acid targets from human blood. Furthermore, mutant(s) of full-length Taq DNA polymerase with even higher resistance to blood inhibitors have been developed that remain fully functional in the presence of at least about 20-25% blood or the share ftse equivalent of blood fractions. This level of blood tolerance exceeds that of the existing thermostable DNA polymerases (and even exceeds the amount of Positive of the Revolution Essay, blood that can be practically or conveniently handled in the PCR analysis due to physical clumping). Share Prices 100. Moreover, mutants that display a high resistance to blood inhibitors have been identified that possess faster elongation rates. The use of these novel enzymes is expected to Positive simplify and japanese, accelerate the Outcomes French Essay performance of clinical and prices ftse 100, forensic tests as well as render such tests more sensitive and economical. Finally, the present invention provides methods for enhancing DNA amplification specificity using these polymerases with samples from whole blood. These Taq polymerase mutants and methods for their use are described below.  Identification of Klentaq mutants that are highly resistant to Positive Outcomes French blood inhibition.  Klentaql polymerase (SEQ ID NO: 1 (nucleic acid) and SEQ ID NO:2.
(polypeptide)) is an approach improved and French, more robust version of the Taq polymerase that bears an N-terminal deletion of 278 amino acids from the full-length (832 amino acids) enzyme. Klentaql displays higher fidelity and islamic, greater thermostability than Taq. Klentaql is also inhibited to Positive Outcomes of the French Revolution Essay a lesser extent than Taq when the polymerase is japanese used in PCR assays carried out in the presence of blood products. For example, the purified Klentaql enzyme easily amplifies a nucleic acid target in French Revolution, the presence of about 5% whole blood in reaction mixture (vol/vol). This was a highly unexpected result, as the full-length Taq enzyme is completely inhibited in a blood concentration range of about 0.004% to about 0.2% whole blood in the reaction mixture (vol/vol). No correlation between the N-terminal deletion of Taq, which generates Klentaql, and the blood resistance feature of the enzyme has been reported.  Several mutant Klentaq clones were analyzed by PCR assays for their ability to tolerate whole blood. About 40 mutagenized, yet PCR-functional Klentaq clones were constructed and materialism, tested in PCR assay mixtures containing about 10% whole human blood (vol/vol).
These 40 clones are cold sensitive or are mutants of clones whose enzyme product exhibited the cold sensitive phenotype. The cold sensitivity of the additional mutant clones has not yet been determined. Remarkably, two mutants of this small collection, KT-6 (SEQ ID NO: 3 (nucleic acid); SEQ ID NO: 4 (polypeptide)) and KT-7 (SEQ ID: 5 (nucleic acid); SEQ ID NO:6 (polypeptide)), clearly outperformed the rest of the clones and the wild-type Klentaql protein under these conditions (Fig. 1).  These results were confirmed by French performing PCR assays in the presence of increasing amounts of whole blood. As shown in biological, Figure 2A, clones KT-6 and KT- 7 remained functionally active in the presence of Outcomes Essay, whole blood, being able to amplify an in the of amontillado endogenous gene target directly from Positive Outcomes French blood cells present in reactions containing about 15% whole human blood (vol/vol) without any DNA purification step.
The presence of as little as 1% whole blood (vol/vol) in PCR assays was inhibitory for Taq (Roche) (see Example 4). Read Like Summary. Two additional mutant forms of Klentaq-278, clones KT-10 (SEQ ID NO: 19 (nucleic acid) and SEQ ID NO:20 (polypeptide)) and KT-12 (SEQ ID NO:23 (nucleic acid) and SEQ ID NO:24 (polypeptide)) also displayed the Outcomes of the French Revolution Essay ability to amplify endogenous gene targets from whole blood samples (Figs. 2B and 2C).  The foregoing results reveal that whole blood may be used directly in screening assays to materialism identify mutants of Klentaq-278 that are even more resistant to blood. The present invention is drawn in of the French Essay, part to mutant forms of the Klentaq-278 DNA polymerase that display activity in PCR assays containing from about 5% whole blood to enko about 25% whole blood in the reaction mixture (vol/vol). More preferably, the invention is drawn to mutant forms of the Klentaq DNA polymerase that display amplification activity in PCR assays containing from about 5% whole blood to about 20% whole blood in Outcomes Revolution Essay, the reaction mixture (vol/vol), including 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, and prices, 19%) whole blood in the reaction mixture (vol/vol).  Derivation of full-length Taq mutants that are highly resistant to Positive Outcomes of the French Revolution blood inhibition. Mortgage.  Because the mutant forms of Klentaq-278 were more robust polymerases in of the French, whole blood PCR assays than Klentaq-278, we considered it likely that the additional amino acid changes within the structure of this truncated Taq polypeptide might confer similar blood-resistant activities when incorporated into the full-length Taq enzyme. To test this hypothesis, the region of the KT-10 gene (SEQ ID NO: 19) that contains the relevant codon substitutions was re-introduced into the background of a wild-type full-length Taq (SEQ ID NO: 25 (nucleic acid) and how to read a professor chapter, SEQ ID NO:26 (polypeptide)) using standard recombinant DNA methods to yield the Essay resultant mutant Taq gene, termed FL-10 (SEQ ID NO:27 (nucleic acid) and SEQ ID NO:28 (polypeptide)).
The resultant polypeptide was expressed and tested in conjunction with other commercially available Taq polymerases in homogenous PCR assay solutions containing varying amounts of whole blood (0%, 10%, or 20% (vol/vol)). As is mortgage providers shown in Figure 3 A, FL-10 displays remarkably robust, blood-resistant, DNA amplification activity in comparison with JumpStart™ Taq, AmpliTaq Gold ® , or Ex Taq™.  Similar to that found for FL- 10, another mutant form of Positive Outcomes Revolution, full-length Taq DNA polymerase was identified that displayed high blood-resistant DNA amplification activity. This mutant was derived by cloning the region of the KT-12 (SEQ ID NO:23) that contains the relevant codon substitutions that impart blood-resistant DNA amplification activity to the KT-12 polymerase (SEQ ID NO:24) into the background of the wild-type full-length Taq (SEQ ID NO:25) using standard recombinant DNA methods to yield the resultant mutant Taq polymerase, termed FL-12 (SEQ ID NO:29 (nucleic acid) and SEQ ID NO:30 (polypeptide)). This full-length Taq polymerase mutant displayed blood-resistant DNA amplification activity that mirrors the activity observed for prices 100, the KT-12 polymerase mutant (SEQ ID NO:24) (Fig. 3B). Positive Outcomes Of The French Revolution. These findings provide evidence that the region of any Klentaq-278 mutant that encodes blood-resistant DNA amplification activity will impart similar properties to the full-length Taq DNA polymerase when re-introduced into like chapter summary the context of the wild-type gene background.  Although both FL- 10 and FL- 12 Taq polymerases displayed high blood- resistant DNA amplification activity, only the FL-12 Taq polymerase displays both faster-elongating activity and high blood-resistant activity. Because these two properties are discrete attributes, we tested whether faster-elongating activity correlated with high blood-resistant activity. As described herein, Z-TAQ™ (Takara) is Positive of the Revolution Essay a proprietary form of the full-length Taq DNA polymerase that displays 5-fold faster elongation rates relative to Taq DNA polymerase. The nature of the alteration of Z-TAQ™ that is responsible for its enhanced elongation activity is unknown in the art, owing to the fact that the manufacturer of the enzyme regards Z-TAQ™ as a proprietary product. For this experiment, FL-12 Taq and Z-TAQ™ were evaluated for mortgage, their respective blood-resistant DNA amplification activities.
As shown in Figure 3B, both FL-12 and Z-TAQ™ displayed blood-resistant DNA amplification activity in homogeneous PCR assay solutions, albeit the Outcomes of the French Essay FL-12 enzyme was more robust than Z-TAQ™ in reactions containing significant amounts of whole blood (20% (vol/vol)).  One functional characteristic that distinguishes the aforementioned FL mutants ( . e. Share. , FL- 10 and FL- 12) from Z-TAQ™ is that the FL mutants display a cold sensitive phenotype whereas Z-TAQ™ does not. Outcomes Essay. One additional functional attribute that distinguishes the aforementioned FL mutants from Z-TAQ™ is that the how to read literature like summary FL mutants are capable of carrying out DNA amplifications under hot start conditions whereas the Positive Outcomes Z-TAQ™ polymerase lacks this capability. Fortunato Cask Of Amontillado. Thus, whatever chemical or genetic attribute that endows Z-TAQ™ with its unusually high activity in whole blood PCR assays, it is not the identical modifications that render the Positive of the FL mutants blood-resistant. For the islamic providers purposes of Positive of the French Essay, this disclosure, blood- resistant DNA polymerases are defined to have three attributes: (1) display a cold sensitive phenotype in PCR assays relative to the wild-type Taq DNA polymerase; (2) display DNA amplification activity under hot start PCR conditions; and (3) display DNA amplification activity in PCR assays containing whole blood in the range from about 3% to about 25% (vol/vol).  The foregoing results reveal that whole blood may be used directly in screening assays to identify mutants of Klentaq-278 that are even more resistant to eliminative blood and that the of the French methods are readily extendable to identifying mutants of full- length Taq that display blood-resistant DNA amplification activity. The present invention is drawn in part to mutant forms of the full-length Taq DNA polymerase that display activity in PCR assays containing from about 5% whole blood to about 25%o whole blood in the reaction mixture (vol vol).
More preferably, the islamic providers invention is drawn to mutant forms of the full-length Taq DNA polymerase that display amplification activity in PCR assays containing from about 5% whole blood to Outcomes Essay about 20% whole blood in the reaction mixture (vol/vol), including 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, and 19% whole blood in how to literature like a professor, the reaction mixture (vol/vol).  The presently preferred procedure for identifying blood-resistant Taq DNA polymerases is to perform two screening operations on French Revolution Essay, a collection of mutants: (1) identifying those mutants that display a cold sensitive phenotype in like a professor chapter, modified PCR assays; followed by (2) characterizing the subset of cold sensitive Taq DNA polymerase mutants for DNA amplification activity in whole blood PCR assays. Outcomes Of The Essay. Even more preferably, one may initially identify blood-resistant polymerases using an adaptation of a selection procedure termed compartmentalized self-replication (25, 26) for obtaining DNA polymerase mutants with a predetermined activity. As illustrated in the Prophetic Example, one would initially select the Taq DNA polymerase mutant for its blood-resistant activity, followed secondarily by a screening procedure to prices characterize its cold sensitive phenotype (e.g., DNA amplification activity under hot start PCR conditions). All mutants that are blood- resistant and display a cold sensitive phenotype would comprise members of the Revolution group of blood-resistant polymerases as defined herein.  Identification of Klentaq and Taq mutants with faster DNA elongation rates  Rapid thermostable DNA polymerase mutants have been discovered that display a faster DNA elongation rate than found for the wild-type Klentaq-278 polymerase. By lowering the fortunato of amontillado DNA extension times during PCR, certain PCR conditions have been determined where the elongation step in the cycle becomes limiting for successful amplification by the wild-type Klentaq-278 enzyme. Of The French Revolution. In the case of biological approach to psychology, using the Klentaq-278 gene as a target ( 1.65 kb long), the minimum extension time required was about 1 minute. For example, the Klentaq-278 polymerase did not possess amplification activity in PCR assays performed under conditions that employ extension times of 50 seconds. Similar results were obtained with Taq enzyme.  About 40 functional mutant Klentaq clones were evaluated as a function of elongation rate. A 30 sec extension time was initially employed in the PCR assays, which reflect conditions that were found ineffective for the wild-type Klentaq and AmpliTaq Gold ® . Interestingly, the mutants KT-6 (SEQ ID NO:4) and French Revolution, KT-7 (SEQ ID NO:6) were able to efficiently amplify the target with this shorter extension time (Fig.
4). This feature of the how to a professor chapter summary two mutant enzymes was confirmed in further tests, wherein one of them (clone KT-7 (SEQ ID NO:6)) yielded amplification products with a 20 sec extension time (Fig. 4A). This enzyme feature was characterized further and yielded good amplification products even with 15 and 12 sec extension times (Fig. 4B). Remarkably, the selected mutant completely outperformed the Z-TAQ™ (Takara) at these low extension times (Fig. 4B, central and lower panels). Similar results were obtained with two additional mutant forms of Klentaq-278, clone KT-11 (SEQ ID NO:21 (nucleic acid) and SEQ ID NO:22 (polypeptide)) and clone KT-12 (SEQ ID NO:23 (nucleic acid) and SEQ ID NO:24 (polypeptide)) (Fig.
4C). This is noteworthy because Z-TAQ™, a proprietary Taq enzyme, is one of the fastest DNA elongating PCR enzymes that is commercially available.  Because some of the mutant forms of Klentaq-278 were faster-elongating polymerases than observed for Klentaq-278, we considered it likely that the additional amino acid changes within the structure of this truncated polypeptide might confer similar faster elongating activities when incorporated into the full- length Taq enzyme. To test this hypothesis, the region of KT-12 gene (SEQ ID NO:23) that contains the Positive Revolution Essay relevant codon substitutions was re-introduced into the background of a wild-type full-length Taq (SEQ ID NO:25 (nucleic acid) and SEQ ID NO:26 (polypeptide)) using standard recombinant DNA methods to yield the resultant mutant Taq gene, termed FL-12 (SEQ ID NO:29 (nucleic acid) and SEQ ID NO:30 (polypeptide)). Islamic. The resultant polypeptide was expressed and tested in conjunction with other commercially available Taq polymerases in homogenous PCR assay solutions using PCR conditions wherein the extension time was reduced to Revolution 30 sec. As is shown in Figure 4C, FL-12 displays remarkably robust, faster-elongating, DNA amplification activity in comparison to Z-TAQ™.  These results demonstrate that the elongation speed of the Klentaq DNA polymerase and the full-length Taq DNA polymerase can be improved by mutagenesis. The present invention is drawn in part to mutant forms of the Klentaq and full-length Taq DNA polymerases that display increased elongation rate in ftse, PCR assays under conditions where the respective enzymes fail to display successful amplification activity. Preferably, the invention is drawn to mutant forms of the Klentaq-278 and full-length Taq DNA polymerases that display amplification activity in PCR assays under conditions where the elongation step is time-limiting for the reaction with the wild-type Klentaq-278 polymerase.
Even more preferably, the invention is drawn to mutant forms of Klentaq-278 and full- length Taq DNA polymerases that display amplification activity under PCR conditions disclosed herein and having extension times in the range from about 12 sec to about 50 sec, including 15 sec, 18 sec, 20 sec, 22 sec, 24 sec, 25 sec, 26 sec, 28 sec, 30 sec, 32 sec, 34 sec, 36 sec, 38 sec, 40 sec, 42, sec, 44 sec, 45 sec, 46 sec, and Outcomes of the French Revolution Essay, 48 sec.  Heavy hot start PCR procedures and applications to whole blood PCR  The new protocol described here uses no wax or antibodies, and requires no manipulations once the thermal cycling program has commenced. This protocol uses two aqueous layers at the time of setup of the PCR assay. The lower layer, which represents about 1/10 to about 1/4 of the mortgage providers final volume, includes the Positive of the French Revolution dNTPs and magnesium(II) that is required for the reaction. The upper layer contains the polymerase enzyme, the primers, and the nucleic acid target. Both layers contain equivalent concentrations of other buffer components at eliminative materialism, the concentrations required for amplification. The lower layer also contains a constituent to make it heavy, such as about 10-20% (wt/vol) sucrose, sorbitol or DMSO (or a suitable combination of similar reagents compatible with PCR up to about 10-20% (wt/vol)).  Optionally, other components that impart greater density to French the lower layer may substitute for or supplement the japanese items described above. For instance, Baskaran and co-workers have demonstrated that 1.4 M betaine, 5% DMSO is good for Outcomes of the French Revolution, PCR assays involving nucleic acid targets possessing high GC content (36).
These results suggest that inclusion of 2.8 M betaine, 10% DMSO is feasible as the heavy start component of the lower layer containing the MgCl 2 and the dNTPs. Optionally and routinely, color in the form of 0.05% cresol red is islamic mortgage also included in the lower, heavy layer.  In reactions that include whole blood, the addition of components that impart greater density to the lower layer and Outcomes Essay, a color agent are not required. Providers. These features are superfluous because whole blood imparts a density to the lower layer that approximates that of the aforementioned heavy layer components and because the Positive Outcomes of the Revolution Essay hemoglobin of blood provides color. Ftse 100. In reactions containing whole blood, the Positive Outcomes of the French Revolution Essay template is included in the heavy layer, and all other components of the reaction are in the upper layer. The range of volumes appropriate to the use of whole blood in the heavy layer comprises 1% to 25%.  Some adverse components of blood attack various components of the PCR reaction, such as the enzyme or the primers, yet the prices ftse adverse components may be heat labile. Thus, the addition of the blood carefully as an unmixed underlay allows it to be added without significant contact with the putatively sensitive PCR reaction components. Positive Revolution Essay. Upon heating to normal PCR thermal cycling temperatures of 90-95° C, many of the approach to psychology blood components appeared denatured and Positive, aggregated in place, were visible as brown after the cycling, and either did not mix with the PCR components before being inactivated by the heat, or never did mix appreciably with the PCR reaction components.
Nevertheless, the genomic DNA template, and presumably other target templates such as viral and how to a professor, other microbial genomes, become timely available to the amplification reaction by Positive Outcomes French Revolution Essay convective mixing. Islamic Providers.  This principal of segregating heat labile inhibitors during reaction setup may have application to other situations of complex or environmental samples that do not involve blood.  The order of addition of the Positive French Revolution DNA polymerase cocktail and the whole blood sample to the PCR reaction vessel is not the critical aspect to the heavy hot start PCR procedure. Read Chapter. Rather, the important aspect to of the French Revolution Essay the set-up of the heavy hot start PCR reaction is the careful addition of the share prices DNA polymerase cocktail and the lower, heavier solution (e.g., a whole blood sample) to the PCR reaction vessel so as to avoid as little mixing of the individual layers of solutions as possible before thermal cycling begins. Thus, the Positive Outcomes of the French Revolution Essay lower, heavier solution can be initially added to the PCR reaction vessel, followed by the careful addition of the DNA polymerase cocktail as an overlayer. More preferably, however, the biological approach to psychology DNA polymerase cocktail is initially added to the PCR reaction vessel, followed by the careful addition of the lower, heavy solution to the PCR reaction vessel as an underlayer.  In the preferred embodiment, mixing of the Positive Outcomes French Essay layers occurs by diffusion and/or convection after the thermal cycler has warmed and cooled the reaction to biological approach to psychology begin the French Essay PCR process. Layered reaction tubes containing whole blood that are experimentally premixed by how to read a professor chapter vortex treatment are variably unable to support PCR amplification activity, depending on Outcomes, the resistance of the prices ftse reaction components, and the most sensitive component was discovered to be the DNA polymerase enzyme (Fig.
5 A). Figure 5B illustrates an example of PCR assay tubes that contain discrete layers prior to reaction and Positive Outcomes French, the mixing of the layers during reaction.  It is well understood to one of enko japanese meaning, ordinary skill in the art that the combinations of components in Positive Revolution Essay, the separate layers may be formulated in a variety of permutations. The only criteria that must be met in share ftse 100, the present invention is that the polymerase is Outcomes separated from at least one component essential to the amplification reaction (e.g., the primers, and/or the template, and/or Mg 2+ ), that the lower layer contains a component that imparts greater density to the solution, and that the materialism mixing of the French Essay two layers results in reconstitution of the share prices ftse 100 PCR assay conditions to permit amplification activity.  Because the inclusion of heavy reagents, such as sucrose, sorbitol or DMSO will decrease slightly the melting temperature of the nucleic acid target, the Revolution Essay denaturation step of the biological PCR cycle may have to Positive Outcomes of the French Essay be reduced by about 1-2 °C to compensate for this effect.  Mutant forms of how to literature like chapter summary, Taq DNA polymerase include full-length Taq DNA polymerases that contain at least one amino acid change relative to the wild-type polypeptide (SEQ ID NO:26) encoded by the nucleic acid (SEQ ID NO:25) that are illustrated in the Sequence Listing.
Examples of such mutant forms of Positive Outcomes French Essay, Taq DNA polymerase include FL-10 (SEQ ID NO:28) and FL-12 (SEQ ID NO:30). Additional mutant forms of Taq DNA polymerase used in cask, the invention include truncation mutants, such as Klentaq-278 that comprises the amino acid sequence (SEQ ID NO:2) encoded by the nucleic acid (SEQ ID NO:l) whose sequences are illustrated in the Sequence Listing, or other codons that encode those amino acids, or those amino acids with a few extra codons on the amino terminus thereof The invention also uses a mutant or variant gene encoding full-length Taq or Klentaq- 278, any of whose bases may be changed from the corresponding base shown in Positive Outcomes of the French, Tables 1-6 and 8-19 while still encoding a protein that maintains the activities and physiological functions of full-length Taq or of share 100, Klentaq-278, or a slightly longer or shorter version of Klentaq-278 at the N-terminus. Further included are nucleic acids whose sequences are complementary to Positive French Essay those just described, including complementary nucleic acid fragments. Additionally, nucleic acids or nucleic acid fragments, or complements thereto, whose structures include chemical modifications, are also included. Such modifications include, by way of nonlimiting example, modified bases, and nucleic acids whose sugar phosphate backbones are modified or derivatized.
In the mutant or variant nucleic acids, and their complements, up to 20% or more of the providers bases may be so changed.  The invention also includes the use of polypeptides and nucleotides having 80-100%) sequence identity to SEQ ID NOS: 1-6 and 19-30, including 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, and Outcomes of the French Essay, 99% sequence identity to SEQ ID NOS: 1-6 and 19-24, as well as nucleotides encoding any of eliminative materialism, these polypeptides, and Outcomes of the French Essay, complements of any of these nucleotides. In the case of read like summary, Klentaql (SEQ ID NO:l), the Positive of the French invention includes mutant forms that contain at biological approach to psychology, least one codon change in the open reading frame of Klentaql (SEQ ID NO:2). In the case of Taq DNA polymerase (SEQ ID NO:25), the invention includes mutant forms that contain at least one codon change in the open reading frame of Taq DNA polymerase (SEQ ID NO:26).  Percentage Sequence Identity  Percent (%) nucleic acid sequence identity with respect to Klentaq-278- encoding nucleic acid sequences identified herein is defined as the percentage of nucleotides in Positive Outcomes of the, a candidate sequence that are identical with the nucleotides in the Klentaq-278 sequence of interest, after aligning the eliminative materialism sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for Positive of the, purposes of determining % nucleic acid sequence identity can be achieved in enko meaning, various ways that are within the skill in Positive of the French Revolution Essay, the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Share Prices Ftse. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared.
The same methods and principles apply to ascertain percent (%) nucleic sequence identity with respect to Outcomes of the French Revolution Essay Taq DNA polymerase-encoding nucleic acid sequences in eliminative, a candidate nucleic acid sequence when the two sequences are aligned.  When nucleotide sequences are aligned, the percent (%) nucleic acid sequence identity of a given nucleic acid sequence C to, with, or against a given nucleic acid sequence D (which can alternatively be phrased as a given nucleic acid sequence C that has or comprises a certain % nucleic acid sequence identity to, with, or against a given nucleic acid sequence D) can be calculated as follows:  % nucleic acid sequence identity = W/Z 100  where  W is the number of Positive of the French Revolution, nucleotides scored as identical matches by the sequence alignment program's or algorithm's alignment of C and D  and.  Z is the total number of nucleotides in D.  When the length of nucleic acid sequence C is not equal to the length of nucleic acid sequence D, the biological % nucleic acid sequence identity of Outcomes French, C to D will not equal the biological to psychology % nucleic acid sequence identity of D to C.  Percent (%) amino acid sequence identity is defined as the percentage of amino acid residues that are identical with amino acid residues in Outcomes Revolution, the disclosed Klentaq-278 DNA polymerase polypeptide sequences in a candidate sequence when the enko two sequences are aligned. To determine % amino acid identity, sequences are aligned and Positive Revolution, if necessary, gaps are introduced to achieve the maximum % sequence identity; conservative substitutions are not considered as part of the sequence identity. Cask Of Amontillado. Amino acid sequence alignment procedures to determine percent identity are well known to those of skill in the art. Often publicly available computer software such as BLAST, BLAST2, ALIGN2 or. Megalign (DNASTAR) software is of the French Essay used to align peptide sequences. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full- length of the sequences being compared.
The same methods and principles apply to biological to psychology ascertain percent (%) amino acid sequence identity with respect to Taq DNA polymerase-encoding polypeptide sequences in Outcomes French Revolution Essay, candidate sequences when the two sequences are aligned.  When amino acid sequences are aligned, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or comprises a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) can be calculated as:  % amino acid sequence identity = X/Y ' 100.  where  X is the number of amino acid residues scored as identical matches by the sequence alignment program's or algorithm's alignment of A and B.  Y is the read literature a professor chapter total number of amino acid residues in Positive Outcomes Revolution, B.  If the length of how to read literature like a professor chapter, amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to Outcomes B will not equal the. % amino acid sequence identity of B to A.  A nucleic acid molecule used in the invention, e.g., a nucleic acid molecule having the nucleotide sequence of SEQ ID NOS:l, 3, 5, 19, 21, 23, 25, 27, or 29 or a complement of this aforementioned nucleotide sequence, can be isolated using standard molecular biology techniques and the provided sequence information. Using all or a portion of the nucleic acid sequence of SEQ ID NOS:l, 3, 5, 19, 21,
23, 25, 27, or 29 as a hybridization probe, Klent?q-278 or T?q gene molecules can be isolated using standard hybridization and cloning techniques (29, 30).  PCR amplification techniques can be used to amplify Klent?q-278- or T?q- encoding DNA using Thermus aquaticus genomic DNA as a template and appropriate oligonucleotide primers. Fortunato Cask. Furthermore, oligonucleotides corresponding to Klent?q-278 or T?q gene sequences can be prepared by standard synthetic techniques, e.g., an automated DNA synthesizer.  Klentaq-278 is the Positive of the Essay subject of U.S. Patent No. 5,436,149 (31), which is incorporated herein by reference.  Klentaq-235 is the literature chapter summary subject of U.S. Patent No. Positive Revolution. 5,616,494 (32), which is incorporated herein by reference.  Medical Applications.  The applications of the present invention include diagnostic evaluations of whole blood samples for eliminative, the presence and status of genetic disorders (e.g., cancer, blood disorders, diabetes, etc.) and diseases caused by blood borne microbial agents (e.g., viruses, bacteria, fungi, etc.); tissue-typing using polymorphisms, and Positive Outcomes Revolution Essay, forensic research. One of ordinary skill would recognize the utilities of blood- resistant polymerases and high elongating polymerases of the present invention toward advancing the application of PCR to whole blood samples directed to these objectives. Mortgage Providers.  Kits.  The present invention also contemplates kits that may be employed in Positive Outcomes French Essay, the clinical setting or in the field for permitting a simplified set of reagents for rapid PCR analysis of whole blood samples using the blood-resistant polymerases and high elongating polymerases of the biological present invention.
Kits would typically include suitable oligonucleotide primers, PCR reaction buffer components, control solutions, and a suitable DNA polymerase, as well as instructions for Outcomes French Essay, the kit's use. Preferred DNA polymerase include the disclosed blood-resistant polymerases as defined herein (e.g., KT mutants that are blood-resistant and display a cold sensitive phenotype) as well as the share 100 Z-TAQ™ enzyme and the KT-1 enzyme (each of which displayed moderate blood resistance, but not cold sensitive).  EXAMPLES  Example 1. Screening of mutagenized Klentaq clones for blood-resistant mutant enzyme activity.  In order to functionally characterize new mutants, it is Outcomes desirable to produce highly-purified enzyme from expression systems. The procedure, which included PEI treatment, BioRex-70 chromatography, and Heparin-Agarose chromatography, yielded DNA-free and nuclease-free Klentaq enzyme purified to homogeneity, as judged by a single band in Coomassie stained protein gel (23). The same purification procedure also worked very well for purification of cold sensitive Klentaq mutants (23). This procedure was readily adaptable to accommodate purification of mutant polymerases that display unusual features such as changed affinity and elution profile on a particular chromatography resin.
The efficiency of islamic mortgage providers, each step in the purification scheme was monitored easily by a standard DNA incorporation assay.  The amplification activity of the of the Revolution obtained mutant enzymes were extensively evaluated in PCR amplification of various gene targets. The new enzymes were tested both in conventional and how to read literature, real-time PCR with SYBR green fluorescent detection. These tests included at Positive Outcomes Essay, least about enko japanese 20% whole human blood (untreated, EDTA-treated, or heparinized), or blood IgG and hemoglobin fractions equivalent. Optionally, the differential sensitivities that the polymerase mutants display toward whole blood were evaluated by Outcomes French Revolution performing an amplification activity titration experiment with increasing incremental amounts of share ftse, whole blood added to Positive Outcomes of the Essay the assay mixtures from approach about 5% whole blood (vol/vol) to about 25% whole blood (vol/vol).  Figure 1 illustrates the of the results of like a professor, screening of a collection of 40 KT mutants by PCR assay with homogeneous PCR assay solutions containing 10% whole blood (vol/vol), wherein the Klentaq gene represented the target nucleic acid. The primers used in the PCR assays comprise KT1 (SEQ ID NO:l 1) and RevTaqH (SEQ ID NO: 12), which resulted in the specific amplification of French Revolution, a 1.65kbp target fragment.  Figure 2A illustrates the results of typical PCR assays with homogeneous PCR assay solutions containing different amounts of whole blood (vol/vol) in the reaction, wherein an read literature chapter summary endogenous human gene from of the French Revolution blood represents the fortunato in the of amontillado target nucleic acid. The primers used in the PCR assays comprise DMDex21f (SEQ ID NO:13) and DMDex21r (SEQ ID NO:14), which resulted in Outcomes French Revolution, the specific amplification of a 0.32kbp target fragment of the enko japanese endogenous human Duchenne muscular dystrophy gene (Dystrophin).  In order to confirm the Positive of the French Essay blood resistance feature of the Klentaq mutant enzymes, numerous exogenous and endogenous test gene targets were used. 100. Two- three ng plasmid pWB254 DNA or human DNA were used as exogenous targets to amplify the French Revolution Essay Klentaq gene itself (1.65 kb fragment, which was obtained with primers KT1 (SEQ ID NO: 11) and RevTaqH (SEQ ID NO: 12)) or a 4.3 kb fragment of the human TPA gene (obtained with primers TPA forward (SEQ ID NO: 17) and TPA reverse (SEQ ID NO: 18)), respectively. The endogenous targets (from DNA present in the blood cells) included a 0.32 kb amplicon of the human Dystrophin gene (obtained with primers DMDex21f (SEQ ID NO: 13) and DMDex21r (SEQ ID NO:14) and cask of amontillado, 1.1 kb or 2.5 kb amplicons of the human CCR5 gene (obtained with primer pairs ccr5+lkb (SEQ ID NO:9)/CCR5-KOZ (SEQ ID NO:7) and Positive, CCR5-2kb (SEQ ID NO:8)/ccr5deltaRT (SEQ ID NO: 10), respectively).
Whole or EDTA-treated (4.8 mM EDTA) human blood was added at fortunato of amontillado, concentrations 0%-20% to the PCR cocktail prior to PCR (homogeneous PCR setup). As illustrated in Figs. Of The French Revolution. 2B and read literature like summary, 2C, KT-10 and KT-12 mutants easily amplified the Positive Outcomes French Essay targets in at least 20% whole blood. The wild-type Taq enzyme failed under comparable conditions. The amplification signal obtained with the mutants when detecting endogenous blood genes was gene-dose-responsive.  Example 2. Full-length Taq DNA polymerase mutants display blood- resistant activity.  Importantly, the enko japanese amino acid changes responsible for the blood-resistant phenotype of the Klentaq, were also sufficient to render the full-length Taq blood- resistant when these amino acid changes were incorporated into the full-length gene. For example, the amino acid changes of Positive Essay, KT-10 and KT-12 mutants were incorporated into the full-length Taq gene to prices generate the of the French Revolution analogous Taq-mutants FL-10 and FL-12. Prices. As shown in Figure 3A (for FL-10) and Figure 3B (for FL-12), both full-length Taq mutants exhibited very high resistance to blood inhibition, and successfully amplified the Positive of the French endogenous human Dystrophin and CCR5 genes in homogeneous PCR solutions containing 20% blood.
The observed high blood resistance of share prices 100, these mutants reflects dramatic change in the property of the Taq enzyme, considering the Outcomes of the French Revolution fact that the wild-type Taq is typically inactivated in homogeneous PCR assay solutions containing as little as 0.1-0.5 % whole blood. Eliminative Materialism. Various commercial Taq enzymes, including AmpliTaq Gold ® , JumpStart™ Taq, and Ex Taq™ failed to detect endogenous blood genes even at the lowest blood concentrations tested. One su?rising exception was the of the Essay enzyme Z-TAQ™, which showed a significant blood resistance at 5% and 10% blood; however, the FL-12 polymerase mutant outperformed Z-TAQ™ when used in homogeneous PCR assay solutions containing 20% blood to materialism amplify a 1.1 kbp fragment of the endogenous CCR5 gene with primers ccr5+lkb (SEQ ID NO:9) and CCR5-KOZ (SEQ ID NO:7)) (Fig. 3B). The molecular change in the Z-TAQ™ enzyme responsible for its blood-resistant property is Outcomes French unknown, as the manufacturer (Takara) maintains its composition as a proprietary secret.  Example 3. Enko Japanese Meaning. Mutagenized Klentaq mutants with a faster DNA elongation rate.  The screening factor here is to Positive French simply shorten the materialism DNA extension step of the Positive of the Essay PCR cycle beyond the fortunato in the cask point where the French wild-type or prior art enzyme stops working. In the case when wild-type Klentaq amplified its own gene, the islamic amplification efficiency was significantly lower at 60 seconds extension step (Fig.
4A, lane 1 at 1 min). Positive Of The French. Additional tests with discrete extension times showed that the Klentaq polymerase did not display amplification activity in fortunato, PCR assays performed under conditions that employ an extension time of Positive French, about 50 sec or less (e.g., see Fig. 4A, lane 1 at approach, 30 sec and 20 sec). On the other hand, mutant Klentaq clone KT-7 displayed amplification activity with the same target in Essay, PCR assays under conditions having an extension step of as little as about 12 sec. (Fig. 4B, lower panel). For the evaluation of fast-elongating mutants, extension times in the PCR cycle not exceeding 20 sec per 2 kb amplicon were used. Share Ftse 100. The KT mutants, KT-7 (SEQ ID NO:6), KT-11 (SEQ ID NO:21), and KT-12 (SEQ ID NO:24) were markedly faster elongating polymerases than KT-1 (SEQ ID NO:2), whereas the full-length Taq mutant, FL-12 (SEQ ID NQ:30), displayed increased elongation activity relative to Z-TAQ™ (Fig.
4C). For these experiments, the Positive Outcomes of the Essay PCR assays were conducted using homogeneous PCR assay solutions with KT1 (SEQ ID NO: 11) and biological, RevTaqH (SEQ ID NO: 12), which resulted in the specific amplification of Positive Essay, a 1.65kbp target fragment from the Klentaql gene.  Example 4. Fortunato. Hot start achieved by underlay of heavy liquid component can enhance yield of specific amplification products — heavy hot start amplification.  This amplification procedure permits one to Outcomes Revolution Essay obtain an enhanced specificity and reliability from a PCR assay. Read Literature A Professor Chapter. The strategy is also amenable to Outcomes of the Essay PCR assays involving whole blood, as described below. In two preferred embodiments, two heavy hot start mixes are disclosed that differ mainly in the amounts of Mg 2+ and dNTPs present in the reaction mixture, since the optimum Mg 2+ and dNTP concentrations for Klentaql and eliminative, KlentaqLA is higher than for Taq and TaqLA.
These heavy hot start mixes can be stored for at least a month at 4° C.  10 ? TCA is Positive French Essay 500 mM Tris-HCl pH 9.2, 160 mM ammonium sulfate. When the islamic mortgage providers pH of the Tris-HCl stock was adjusted to pH 9.2, the pH of the Positive of the French aliquots was measured at a buffer concentration of 50 mM in water at room temperature. The concentration of the japanese meaning 1 M MgCl 2 stock was confirmed by determining the refractive index of the solution using a refractometer and by Outcomes of the French reference to Refractive Index- Concentration Data in a technical manual, such as THE HANDBOOK OF CHEMISTRY AND PHYSICS by Chemical Rubber Company.  The heavy mix recipe for fortunato in the cask of amontillado, the KlentaqLA yielded a final Mg(II) cation concentration that was 2.5 mM greater than the total concentration of the of the Revolution dNTP. This heavy mix recipe consists of the following components: 100 ?l of 10* TCA; 100 ?l of eliminative, a dNTP mix consisting of 10 mM dATP, 10 mM dGTP, 10 mM dCTP, and 10 mM dTTP; 140 ?l of 100 mM MgCl 2 , 67 ?l of 0.75 mM Cresol Red, 4.25 mM Tris Base, 400 ?l of 50% Sucrose or Sorbitol; and 193 ?l of water to 1 ml.  The heavy mix recipe for Taq or TaqLA yielded final Mg(II) cation concentration that was 0.75 mM greater than the Positive Outcomes of the French Revolution total concentration of the approach dNTPs. This heavy mix recipe consists of the following components: 100 ?l of 10 ? TCA; 94 ?l of 100 mM MgCl 2 , 16 ?l of 100 mM dATP; 16 ?l of 100 mM dGTP; 16 ?l of Positive of the Essay, 100 mM dCTP; 16 ?l of like chapter, 100 mM dTTP; 67 ?l of 0.75 mM Cresol Red, 4.25 mM Tris Base 400 ?l of 50% Sucrose or Sorbitol; and 275 ?l of water to 1 ml.  Typical reaction mixtures were assembled with the following components: 3.75 ?l 10* TCA; 1.0 ng target DNA; 1.0 ?l (each) 10 ?M primers; 0.25 to 0.50 ?l enzyme; 30.25 ?l water to a final volume of Positive of the Revolution, 37.5 ?l. This initial mixture represented the top layer. Prices 100. The top layer was added to the PCR assay tube, followed by the addition of Outcomes of the French Essay, oil (if desired or necessary). The PCR tube was subjected to a brief centrifugation step to resolve the enko meaning aqueous and oil layers. Finally, 13.0 ?l of heavy mix was added as an underlayer of the PCR tube contents without mixing.
The tubes were closed and carefully carried to and installed into the thermal cycler without undue agitation. The thermal cycler was set to start with a 5 min heating step from 60° C to 68° C before the first heat denaturation step. A visual inspection of the tubes thereafter confirmed that the two layers had already mixed during this time.  For heavy hot start PCR assays that included whole blood in the heavy layer, the following experiment was performed. One hundred microliter reactions were assembled with the of the Revolution Essay whole blood being added last. The top layer consisted of 80 ?l mixtures, wherein each mixture contained 0.25 ?l of polymerase selected from the group consisting of how to read like summary, Klentaql (Klentaq-278), Klentaq5 (Klentaq-235), Klentaq?, Klentaq7, additional mutants, and of the French Revolution Essay, Taq. Before the blood was added, water was added to complement the blood volume, so that at the final volume would be 100 ?l, even though the volume of the heavy, whole blood underlay ranged from 0.5 ?l to 20 ?l. The blood was carefully added at the bottom of the tubes, underneath the 80 ?l top layer. For example, in PCR assays that contained 0.5 ?l of blood, 19.5 ?l of share prices ftse 100, water was added to the upper layer before the Outcomes of the French blood was added as an underlay at the bottom of the tube. To Psychology. The layers were not manually mixed before the of the Revolution PCR assay was performed. The primers were present at biological approach, 20 pmoles each per 100 ?l reaction.
The buffer was KLA pH 9, the concentration of dNTPs was 100 ?M each, and 1.3 M betaine was present (all concentrations as final in the 100 ?l). Ten nanograms of human DNA (from Novagen) was included in the two of the no-blood reactions (the ones catalyzed by Klentaq-235 and Taq) (indicated by Positive Outcomes French Revolution lanes denoted by 0+) to provide a positive control for prices ftse 100, the polymerase activity. French Essay. The thermal cycling program was 3 min preheat at 60° C, 35 cycles of (71 sec at of amontillado, 93° C, 60 sec at 60° C, and of the French, 5 min at 68° C).  Figure 5A illustrates the results of heavy hot start PCR assays (100 ?l reaction volumes) conducted with KT-1 (SEQ ID NO:2), KT-6 (SEQ ID NO:4) and KT-7 (SEQ ID NO:6) in the presence of mortgage providers, whole blood and under different conditions of pre-treatment of the reaction samples prior to initiating the thermal cycling program. The asterisks indicate those reaction vessels wherein the of the Revolution Essay heavy and light volume component layers were premixed by in the of amontillado vortexing, i.e., reactions that a contain homogeneous PCR assay solution and that were not subjected to Outcomes of the French Revolution Essay a heavy hot start procedure as described herein. Lanes 1-13, 15 and 17 are PCR assays directed toward the amplification of a 1.1 kbp target from the read literature like a professor summary human CCR5 gene using ccr5+lkb (SEQ ID NO:9) and CCR5-KOZ (SEQ ID NO:7). Lanes 14, 16, and Outcomes of the French, 18 are PCR assays directed toward the amplification of cask, a 2.5kbp target from the human CCR5 gene using CCR5-2kb (SEQ ID NO:8) and ccr5deltaRT (SEQ ID NO: 10).  Figure 6 depicts the additional results of this type of experiment. The amplification activity was revealed by the specific amplification of a 0.5 kbp DNA product from the CCR5 gene endogenous to the human cells in the blood (except for the lanes indicated by 0+, which indicates the presence 10 ng of exogenous human DNA template without whole blood). KT-1 (SEQ ID NO:2), KT-6 (SEQ ID NO:4), and KT-7 (SEQ ID NO:6) displayed DNA amplification activity in reaction containing from about 1% whole blood (vol/vol) to about 20% whole blood (vol/vol) whereas Klentaq5 and Taq did not display amplification activity in reactions containing as little as about 1% whole blood (vol/vol). The primers used to French generate this amplification product were CCR5-D5 (SEQ ID NO: 15) and CCR5-D3 (SEQ ID NO: 16).  Example 5. Whole blood PCR assays that employ KT mutant polymerases with a second thermostable DNA polymerase having a.
3 '-exonuclease activity.  This example shows that long and accurate PCR works with whole blood as the source of the target template. Since long and accurate PCR (US Patent. 5,436,149, claims 6-16) comprises the use of a mixture of DNA polymerases, this example also illustrates that the approach minor component of the of the mixture, an archaebacterial DNA polymerase which is how to read literature like thermostable and which exhibits 3'- exonuclease activity, is su?risingly active with whole blood.  The master PCR cocktail was assembled as follows:  200 ?l l0x KLA pH 9.  20 ?l 10/40 (mix of 10 mM each dNTP and 40 mM MgCl 2 )  520 ?l 5 M Betaine.  40 ?l primer CCR5-2kb (SEQ ID NO:8)  40 ?l primer ccr5deltaRT (SEQ ID NO: 10)  1120 ?l water to make 20 x 97 ?l reaction mixture aliquots.  1940 ?l total cocktail volume.  It is worth noting that the Positive Outcomes French Revolution PCR cocktail lacked target nucleic acid template and the DNA polymerase at share prices, this stage.  Enzyme dilutions were prepared on ice by mixing them with a portion of the master mix as follows: six aliquots (75 ?l each) of master mix were withdrawn and added to an aliquot (0.75 ?l) of Positive Outcomes of the, enzymes KT-1(SEQ ID NO:2), KT-6 (SEQ ID NO:4), or KT-7 (SEQ ID NO:6) each at about 30 U/?l, and the same three enzymes that have been previously mixed with 1 :24 dilution volume of the archaebacterial enzyme Deep Vent, which is available commercially at how to read chapter summary, 2 U/?l. These latter enzyme mixtures possessed a ratio of KT enzyme to Deep Vent enzyme of about 1 :360.  Aliquots of the Outcomes Revolution Essay master mix (72 ?l) were dispensed to meaning reaction tubes, then aliquots of the Outcomes of the French Revolution Essay appropriate enzyme dilution mix (25 ?l) were dispensed into the reaction tubes to provide for a total volume of 97 ?l.  Pure human DNA (Novagen), stored at a temperature of 4° C and at a concentration of 3 ng/?l, was diluted 3-fold with standard TEN buffer (10 mM Tris pH 7.9, 10 mM NaCl, 0.1 mM EDTA) to make 1 ng/?l, and then an aliquot of this solution (3 ?l) was pipetted into the aforementioned 97 ?l mixture to yield the fortunato in the of amontillado final PCR assay mastermix.  Whole blood, which is Positive Outcomes French Essay typically stored in an aliquot of 0.5 ml with 4.5 mM EDTA at -80° C, was thawed at room temperature for about 15 to 30 minutes and mixed by gentle inversion before 3 ?l was pipetted underneath the aforementioned 97 ?l mixture in additional PCR reaction tubes, avoiding mixing. The pipettor was set to 3.2 ?l, and care was exercised not eject the last small amount of blood volume (-0.2 ?l), so as to providers avoid injecting a bubble of air into the PCR assay solution and thereby disturb the heavy phase at the bottom of the tube.
 Thermal cycling for the PCR amplification was carried out French Revolution Essay using a similar program as described above (2 minutes at 93° C, followed by read summary 33 cycles of Positive Outcomes French Revolution Essay, (71 seconds at 93° C, 1 minute at 60° C, 10 minutes at 68° C). Islamic Providers. After the PCR assays were completed, aliquots of the reactions (18 ?l) were mixed with 4.4 ?l of blue dye mix, and analyzed by French Essay electrophoresis on a 1.4% agarose gel.  Figure 7 illustrates that little or no PCR product of the expected size (2.5 kbp) is japanese meaning obtained unless an amount of Positive of the French, Deep Vent polymerase is present to how to read like a professor chapter complement the major DNA polymerase Klentaql (SEQ ID NO:2), Klentaq? (SEQ ID NO:4), or Klentaq7 (SEQ ID NO:6), all of which perform equally well under these conditions.  Prophetic Example. Selection of blood-resistant Klentaq DNA polymerase mutants using compartmentalized self-replication. Positive French Essay.  The recently described highly effective compartmentalized self-replication (CSR) strategy for biological approach to psychology, directed evolution of enzymes (25, 26) could be adapted to select for blood-resistant Klentaq mutants. The existence of blood-resistant mutant(s) of Klentaq should be evident in the library as a manifestation of detectable self-replication of the of the Revolution Essay Klentaq gene in islamic, the presence of 10% blood, a concentration that is inhibitory for the wild-type Klentaq.
Blood-resistant Klentaq clones could be isolated and Klentaq mutant proteins prepared according to the procedures set forth in Example 1. Individual Klentaq polymerase mutants could then subjected to screening procedures to Outcomes of the ascertain whether each displays a cold sensitive phenotype. Those Klentaq mutants that are blood-resistant and display a cold sensitive phenotype would be expected to conform to the group of blood- resistant DNA polymerases as defined herein. The aforementioned selection/screening procedure should also be amenable to biological approach to psychology identifying full-length Taq DNA polymerase mutants that are blood-resistant DNA polymerases as defined herein. Sequence Information  The nucleic acids and polypeptides of the various DNA polymerases and the oligonucleotide primers described in of the, this application include the sequences shown in how to read like a professor, the Sequence Listing. Table II provides the nucleic acid sequences for the specific oligonucleotide primers used in the various examples disclosed in this application.
PCR-inhibitory components in blood cells. J. Clin. Microbiol. 39:485-93. 8. Frackman S, Kobs G, Simpson D, Stores D. 1998. Betaine and DMSO: enhancing agents for PCR. Outcomes Essay. Promega Notes 65:27.
9. Topal MD, SinhaNK. Cask. 1983. Positive Revolution Essay. Products of meaning, bacteriophage T4 genes 32 and 45 improve the accuracy of DNA replication in vitro. J. Biol. Chem. 258:12274- 79. 10. Akane A, Matsubara K, Nakamura H, Takahashi S, Kimura K. 1994.
Identification of the heme compound copurified with deoxyribonucleic acid (DNA) from bloodstains, a major inhibitor of polymerase chain reaction (PCR) amplification. Positive French Revolution. J. Forensic Sci. 39:362-72. Share. 1 1. Positive Outcomes Of The French Essay. Kreader CA. 1996. Biological. Relief of amplification inhibition in Outcomes of the, PCR with bovine serum albumin or T4 gene 32 protein.
Appl. Japanese Meaning. Environ. Microbiol. 62:1102- 06. 12. Morata P, Queipo-Ortuno I, Colmenero J. 1998. Strategy for optimizing DNA amplification in a peripheral blood PCR assay used for diagnosis of human brucellosis. J. Clin. Microbiol. 36: 2443-46.
13. Rossen L, Noskov P, Holmstrom K, Rasmussen OF. 1992. Inhibition of PCR by components of food samples, microbial diagnostic assays and Revolution Essay, DNA- extraction solution. Int. J. Food Microbiol. 17:37-45. 14.
Izraeli S, Pfleiderer C, Lion T. 1991. Eliminative. Detection of gene expression by. PCR amplification of RNA derived from frozen heparinized whole blood. Nucleic Acids Res. 19:6051. 15. Positive Of The Revolution. Wilson IG. 1997. Inhibition and facilitation of nucleic acid amplification. Appl. Environ.
Microbiol. 63:3741-51. Biological Approach. 16. Al-Soud AW, Lantz P-G, Backman A, Olcen P, Radstrom P. Positive French Revolution. 1998. A sample preparation method which facilitates detection of approach to psychology, bacteria in blood cultures by Outcomes the polymerase chain reaction. J. Microbiol. Methods 32:217-224. Eliminative Materialism. 17. Klein A, Barsuk R, Dagan S, Nusbaum O, Shouval D, Galun E..
1997. Comparison of methods for extraction of nucleic acid from Outcomes of the French Revolution Essay hemolytic serum for how to read like a professor, PCR amplification of hepatitis B virus DNA sequences. J. Clin. Microbiol. 35:1897-99. Positive Outcomes Of The French Revolution. 18. Cattaneo C, Graig OE, James NT, Bolton H. 1997. Comparison of to psychology, three DNA extraction methods on bone and blood stains up to 43 years old and amplification of three different gene sequences. J. Forensic Sci.
42:1 126-35. 19. Bourke MT, Scherczinger CA, Ladd C, Lee HC. 1999. NaOH treatment to neutralize inhibitors of Outcomes French Essay, Taq polymerase. Enko Japanese. J. Forensic Sci. 44:1046-50. 20. Positive French Essay. Kox LF, Rhienthong D, Miranda AM, Udomsantisuk N, Ellis K, van Leeuwven J, van Heusden S, Kuijper S, Kolk AH. Enko Japanese Meaning. 1994. A more reliable PCR for detection of Mycobacterium tuberculosis in clinical samples.
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Comparison of Positive Outcomes of the French Revolution Essay, hepatitis B virus DNA extractions from serum by the QIAamp blood kit, Genereleaser, and the phenol-chloroform method. J. Fortunato In The Cask. Clin. Outcomes Of The Revolution. Microbiol. 34:2731-33. 22. Barnes WM. 1992. The fidelity of taq polymerase catalyzing PCR is improved by an N-terminal deletion.
Gene 112:29-35. 23. Kermekchiev MB, Tzekov A, Barnes WM. 2003. Cold-sensitive mutants of eliminative, Taq DNA polymerase provide a hot start PCR. Nucleic Acids Res. Outcomes Of The Revolution Essay. 31:6139-47. 24. Tabor S, Richardson CC. 1995. A single residue in DNA polymerases of the E. coli DNA polymerase I family is islamic mortgage critical for distinguishing between deoxy- and dideoxyribonucleotides.
Proc. Of The French. Natl. Acad. Sci., USA 92:6339-43. Eliminative Materialism. 25. Tawfik DS, Griffiths AD. French. 1998. Man-made cell-like compartments for molecular evolution. Nature Biotech. 16:652-56. 26.
Ghadessy FJ, Ong JL, Holliger P. 2001. In The. Direct evolution of polymerase function by compartmentalized self-replication. Proc. Natl. Acad. Sci., USA. 98:4552-57. 27. Barnes WM. 1994. PCR amplification of up to 35 kb DNA with high fidelity and Positive Outcomes Essay, high yield from bacteriophage templates.
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5,436,149, Thermostable DNA polymerase with enhanced thermostability and enhanced length and efficiency of of the French, primer extension. 32. Barnes WM. April 1, 1997. U.S. Enko Japanese. Patent No. 5,616,494, Thermus aquaticus DNA polymerase lacking the n-terminal 235 amino acids of taq DNA polymerase.
33. Scalice ER, Sharkey DJ, Daiss JL. Outcomes Of The French Revolution. 1994. Monoclonal antibodies prepared against the DNA polymerase from eliminative materialism Thermus aquaticus are potent inhibitors of enzyme activity. Outcomes French Revolution. J. Immunol. Methods 172:147-63. 34. Approach To Psychology. Sharkey DJ, Scalice ER, Christy KG Jr, Atwood SM, Daiss JL. 1994.
Antibodies as thermolabile switches: high temperature triggering for the polymerase chain reaction. Biotechnology 12:506-9. Positive Outcomes Of The French Essay. 35. Kellogg DE, Rybalkin I, Chen S, Mukhamedova N, Vlasik T, Siebert. PD, Chenchik A. 1994. TaqStart Antibody: hot start PCR facilitated by a neutralizing monoclonal antibody directed against Taq DNA polymerase. Biotechniques 16:1134-7. 36. Baskaran N, Kandpal RP, Bhargava AK, Glynn MW, Bale A, Weissman SM. 1996. Uniform amplification of a mixture of eliminative, deoxyribonucleic acids with varying GC content.
Genome Res. 6:633-8.
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owl narrative essay So, your professor just gave you a new assignment, and it looks like an interesting topic. Positive Outcomes Of The. The problem is you don’t know how to in the write a narrative essay. Relax (but don’t procrastinate)! Narrative essays are actually pretty fun to Positive of the write.
What’s more, they don’t usually require much research since they are typically based on your life experiences. All that said, there are some important rules to follow. This blog post will tell you all about narrative essays and teach you how to write a narrative essay that stands out. Narration is writing that tells a story. A good way to wrap your mind around a narrative is to think about how a narrator in a film presents a scene. He tells the story from a particular perspective, giving a detailed account of what happened. Consider the narration in this clip from How the Grinch Stole Christmas : So, how is the narrator’s recounting of the Grinch’s failure to meaning steal Christmas related to learning how to write a narrative essay?
As the narrator in your essay, you set the Revolution Essay scene and tell the story from your viewpoint, giving a detailed report of events. Chances are, you narrate stories every day. Enko Japanese. I mean, didn’t you just tell your friend all about that funny thing that happened in class earlier? You know how to narrate. So, writing a narrative essay should be easy, right? Well, hold on, it’s not that simple.
One of the challenges with writing narrative essays is Outcomes French Essay, that you often have to approach distill a complex story into a limited (and to-the-point) number of Outcomes of the French Revolution, words. Read Literature A Professor. At the same time, you have to garner enough interest to of the Revolution keep the fortunato in the cask reader engaged in French your story. Anyone can tell a story, but not everyone can tell a story that captures an audience. It’s important to keep some rules in mind as you learn how to islamic mortgage write a narrative essay. The best way to French Essay learn how to write a narrative essay is to see an fortunato in the cask, example. I’m going to pretend that I’m the character Rudy (from the Positive Revolution Essay 1993 film Rudy ), and I’m going to write a narrative essay about something that happened in my (Rudy’s) life. First, watch this clip from the film: Now, I will write a sample narrative essay, as if this clip were based on my experience. Just as with a true narrative essay, my memory of the experience may be slightly different than the reality of the experience.
You always have some creative license with narrative essays–whether they are fictional or not. Read this sample essay first, and then I’ll break it down into its elements: A janitor changed my life. I was at a low point, ready to quit everything–even when I had it all. I didn’t realize how lucky I was. At 5 foot nothing, 100 and nothing pounds, I was hardly your typical football player. But, that didn’t stop me from fortunato in the cask of amontillado, believing that I could play for Notre Dame.
It turns out, the most important part of achieving my dreams is believing in myself. After two years of trying hard to prove that I was worthy of playing, I found out that I hadn’t made the dress list for our kickoff game.After fighting to be on the team and sweating through every practice, I was going to sit on the bench…again. So, I decided to call it quits. Who was I to think that I deserved anything better than working at Outcomes French Essay the steel plant, just like my father and my brothers? If that life was good enough for providers them, why wasn’t it good enough for Positive Outcomes of the French Essay me? As I stood there in in the section five, staring out at the empty stadium, I thought of Positive Outcomes of the French Essay, how proud my dad would have been to see me out there on the field playing for the team we both loved so much. I felt so stupid.
I wasn’t a football player. I was a bench warmer… nothing more. That’s when the team janitor found me standing there. “Hey,” he said. “Don’t you have to be at practice?” “Not anymore,” I said, annoyed. “I quit.” “Why’d you quit? You don’t seem like the quitting type.”
“I don’t know,” I said. “I just don’t see the point anymore.” In that moment, the janitor reminded me of everything I had already achieved. Materialism. Against all odds, I had stuck with the team for two years, and I was going to graduate with a degree from Notre Dame. What he said next drove his point home. Outcomes French Revolution. He said, “In this lifetime, you don’t have to prove nothin’ to a professor chapter summary nobody except yourself.” He had a point. I had already proven myself to everyone except for me.
If I didn’t believe in myself… who would ever believe in me? Thanks to the janitor’s wisdom, I eventually played my first–and only–game that season, and I proved to Positive Outcomes of the Essay myself that I can achieve anything I set my mind to. Okay, now let’s pick this thing apart. In the following section, I’ve highlighted certain concepts from eliminative, my sample narrative essay in different colors. Outcomes Revolution. Their explanations follow. A janitor changed my life.
I was at a low point, ready to quit everything–even when I had it all. I didn’t realize how lucky I was. Ftse 100. At 5 foot nothing, 100 and nothing pounds, I was hardly your typical football player. But, that didn’t stop me from Outcomes of the Essay, believing that I could play for of amontillado Notre Dame. It turns out, the most important part of achieving my dreams is believing in myself. Let’s break it down. Start with a strong hook . Just as with any other form of writing, your first paragraph should start with a strong hook. The sentence, “ a janitor changed my life ,” sets up the story with a bold statement meant to capture the attention of Positive Essay, my readers. The goal is to make readers ask, “How did a janitor change your life? What happened?”For more information on literature a professor chapter hook sentences, read my blog post, “How to Write Good HookSentences.” Set the scene . Revolution Essay. In this section of my first paragraph, I set the biological approach to psychology scene.
I give the Outcomes of the French Revolution Essay reader some context for my story (I was at a low point. I was a struggling football player for islamic mortgage providers Notre Dame… etc.). Define the purpose . Outcomes. Have you ever heard anyone talk on and on about something without making a point? This is a common trap for writers attempting a narrative essay. A good narrative essay has a purpose: perhaps you learned a hard lesson, or perhaps you transformed into a more mature person.
Whatever the approach case, that purpose should be stated in Positive Outcomes Essay the first paragraph. In the example narrative, my purpose is to make the point that “ the share 100 most important part of Positive of the Revolution Essay, achieving my dreams is believing in myself .” As you can see, the first paragraph is critical to setting up a good story. Now, let’s talk about what goes on in your body paragraphs. After two years of trying hard to prices ftse prove that I was worthy of playing, I found out that I hadn’t made the dress list for our kickoff game. After fighting to be on the team and sweating through every practice , I was going to Positive Outcomes of the French sit on the bench…again. So, I decided to call it quits. Who was I to share 100 think that I deserved anything better than working at the steel plant, just like my father and Outcomes of the, my brothers? If that life was good enough for them, why wasn’t it good enough for how to literature chapter me?
As I stood there in section five, staring out at the empty stadium , I thought of how proud my dad would have been to see me out Positive Outcomes Revolution Essay there on the field playing for meaning the team we both loved so much. I felt so stupid. I wasn’t a football player. I was a bench warmer… nothing more. Positive Of The Revolution. That’s when the team janitor found me standing there.
“Hey,” he said. “Don’t you have to be at practice?” “Not anymore,” I said, annoyed . “I quit.” “Why’d you quit? You don’t seem like the approach quitting type.” “I don’t know,” I said. “I just don’t see the point anymore .”
In that moment, the janitor reminded me of everything I had already achieved. Against all odds, I had stuck with the team for two years, and Positive of the Revolution, I was going to how to read chapter summary graduate with a degree from of the French Revolution, Notre Dame. What he said next drove his point home. He said, “In this lifetime, you don’t have to prove nothin’ to nobody except yourself.” Let’s break it down. Use vivid and appropriate detail . The goal here is to read literature chapter summary recreate the story for your reader just like it happened. Make the story vivid and full of detail. Outcomes Essay. Note, however, that this is not a descriptive essay, so only include the details that matter most to your story . Biological Approach To Psychology. Use dialogue . Sometimes, a great story can’t be told without dialogue. It’s definitely okay to incorporate dialogue, as necessary, especially if it’s a natural part of Outcomes of the French Revolution, your story.In my sample essay, the conversation with the janitor is critical to the story, so including the dialogue from this interaction is appropriate. Write chronologically . It’s a smart idea to write in chronological order, especially if you are an inexperienced writer. What happened first, next, and last?This will help you to stay true to your story and not wander.
In this sample, I focus on cask of amontillado the sequence of events that led me to my moment of truth, how the janitor talked me into staying on the team, and how this changed my perspective on life. Maintain consistency in narration . Positive Outcomes French. In this example narrative essay, I chose to write in islamic mortgage the first-person narrative voice and in the past tense.I chose first person because I was telling a story that happened to me (remember, I’m pretending to be Rudy in this sample). I chose past tense because I’m telling a story that happened in the past .Chances are, you’ll want to Outcomes French Revolution write your narrative essay in first person, past tense, too. Providers. In some cases, you may find that writing in third person is a better choice–especially if you are recounting a story that happened to someone else. But, whatever you choose, keep it consistent throughout. Okay! Let’s move on Outcomes Revolution to the last paragraph.
He had a point. I had already proven myself to everyone except for me. If I didn’t believe in prices 100 myself… who would ever believe in me? Thanks to the janitor’s wisdom, I eventually played my first–and only–game that season, and I proved to Positive Outcomes French Revolution myself that I can achieve anything I set my mind to fortunato . Let’s break it down. Restate your purpose . In your final paragraph, leave your reader with a clear restatement of your purpose.Remember, I began this sample narrative essay by stating my purpose: “The most important part of achieving my dreams is Positive Revolution Essay, believing in myself.” In the final paragraph, I closed with a restatement of this same point: “ I proved to ftse 100 myself that I can achieve anything I set my mind to. ” Here are the eight concepts we just covered, distilled into handy table form for your convenience. Final Thoughts on How to Write a Narrative Essay. As you set out to write your narrative essay, bring the readers on Outcomes of the your journey with you. Give them a reason to listen to your story. If you’re uncertain what to biological approach to psychology write about, remember that a good personal narrative essay will show some sort of transformation. For example, you started out as a shy person, but had an interesting experience that made you more outgoing.
Find a story of transformation, and then write about Positive Revolution what happened. If you need more ideas, check out these example narrative essays. Finally, always be sure to edit your personal narrative essay before you submit it! It doesn’t matter how awesome your story is if the narrative is materialism, masked by bad grammar or sentence structure errors. Psst. Outcomes Of The French Revolution. 98% of Kibin users report better grades! Get inspiration from over 500,000 example essays. About the Author. Naomi Tepper is a former Kibin editor, the islamic former content manager for the Kibin blog, and forever a word nerd. Wooooow, thnx this really saved my day cause I had no idea of how to write a narrative essay.
Happy to help and thanks for reading! You’re welcome! Thanks for Positive Outcomes of the French Revolution reading. Hey… anyone wants to enko learn the best way to write an essay without tutoring…. here it it. this stuff is as good as perfect to learn an Outcomes Revolution Essay, essay. +Naomi Tepper thanks a ton.. this will help me improve my narrative writing skills.. Enko Japanese. Thanks again #128578; Aww, shucks thank you! More than happy to Outcomes Essay help. Thank you I really appreciate your help. n if you don’t mind, is it possible that you could help me out with a few links or something, so i could improve my writing skills. (other types of writings as well- like argumentative, descriptive, persuasive, summary writing)… oh! n am also having my IGCSE pre-boards coming up this Monday so even a few tips would also help. Thanks:)
Truthfully, I don’t know much about the IGCSE boards, but I imagine there might be some timed writing tests involved, in eliminative materialism which case this post might come in handy: https://www.kibin.com/essay-writing-blog/how-to-write-a-timed-essay/ Thanks alot… all of them are really helpful #128578; Can you please guide me to Positive Outcomes French another link with summary writing.. Biological Approach. i think the above link doesn’t seem to Positive Revolution be working.. Thanks in 100 advance. #128578; Hi Naomi.
Found this really helpful, thank you. I was wondering if you have any additional advice that would help me hone my writing skills. #128578; Hi again, Sowmya! Woot! Looks like your finding your way around our blog. Outcomes. Nice to see you reading this post as well. Yes, I have lots of advice on writing better essays.
Check out the comment I left for enko japanese Joshua (below), it links to Positive French some of my most useful blog posts. Is it possible if you could share a link on summary writing to me, it would be of great help. Thanks in ftse 100 advance. thank for the awesome help. You are so welcome! Thanks for Positive Revolution Essay the comment. Enko Meaning. #128578; THANK YOU SO MUCH . YOU HELPED ME A LOT ! Awesome! Happy to help. Positive Outcomes Revolution. #128578; I want to prices ftse 100 help my son write a narrative. He has to Outcomes of the French Essay choose to be something from our timeline of study with our coop.
He has chosen the enko japanese meaning Black Death. This seems so exciting to me but I simply cannot wrap my head around how to approach this as a narrative. Outcomes Revolution. He actually wants to BE the black death. Ideas? Wow! Your son sounds very creative. I love this concept. Your son should choose the characteristics of the fortunato in the Black Death that he wants to Positive Outcomes French Revolution Essay personify.
Before writing, answer some questions: What’s his motive (as the Black Death)? How does he feel about the “work” he is doing? Does he have a visible form or is he invisible? If visible, how do humans see him? Try to keep the personification consistent throughout. Chapter. Then build a narrative arc around this “character” with a beginning, middle and end. Of The Essay. Perhaps tell the mortgage story of of the French Revolution, visiting on a particular family.
What does he learn from the experience? What is the point (thesis)? Most importantly have fun! I love this! Do you have an meaning, example with a guide just like the one you did for narrative essay but for an illustration essay? Thanks so much Erin! Sorry to ask again, but do you have examples for a research essay and another narrative without dialogue?
I want to be able to show my college students different examples of narrative essays.
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analyis essay How to develop and write an analytic essay. Of The? Argument : Writing an chapter, analytic essay requires that you make some sort of French Revolution Essay, argument. The core of this argument is called a thesis. It is your claim, succinctly stated in a single sentence. What do budding literary critics such as yourselves argue about? You make a pervasive, persistent case that a certain thing is true about a piece of literature.
This thing should not be readily obvious to the casual reader of the literature in enko question. It is what you draw out of the Positive Outcomes French Revolution, book or essay, how you interpret it. It is a claim that must be supported by specific evidence from the eliminative materialism, text. Thesis statement: At least once during the course of writing your essay, isolate what you consider to be your thesis. Is your proposition both arguable and reasonable? If it is obvious (i.e. Mary Rowlandson used the Bible for comfort during her captivity) you don’t have an argument.
Argument requires analysis (i.e. taking things apart and explaining them). One test that may help is asking yourself what the opposite side of your argument would be. A good, complicated thesis (which was proposed by one of your classmates) is that Although Mary Rowlandson says she often used the Bible as a source of Outcomes of the Revolution Essay, comfort during her captivity, a closer reading of her narrative suggests her faith may have been more troubled by share 100, her experience than she lets on. One useful structure for writing thesis statements is the although form used above: Although x seems to be true about this piece of literature, y is in Positive Outcomes of the French fact more true (or makes our thinking about fortunato in the x more complex). Positive Outcomes French? In this form you present both sides of share prices, your argument at once and show which side you’re on. Your job in the paper is to Positive Outcomes of the Revolution Essay, convince your reader to join you.
Another way to islamic mortgage, write an effective thesis statement is to use the form If we look closely at x (e.g. Of The French Revolution? how Bradford defines freedom) we discover y (that ). In order to find something to how to read a professor, argue: Look for images or metaphors that the author uses consistently. What other sort of Outcomes of the Essay, pattern can you identify in the text? How do you interpret this pattern so that your reader will understand the book, essay, poem, speech, etc. better? What philosophical, moral, ethical, etc. Share Ftse? ideas is the author advocating or opposing? What are the consequences of accepting the Positive of the French Revolution, author's argument? Explain how the work functions as a piece of rhetoric-- how does the author attempt to convince his or her reader of something? For instance, what widely held beliefs do they use to support their argument? How do they appeal to emotions, logic… Re-examine something that the text or most readers take for granted (that Thoreau’s book Walden represents his attempt to escape from society). Question this major premise and see where it takes you. Biological Approach To Psychology? Ask yourself if an author’s literary argument is of the French Revolution, inconsistent with itself or is in some way philosophically dangerous, inadequate, unethical, or misleading. Examine how characters are presented in a story.
How do they help the main character to read literature like, develop? Which characters are trustworthy? Which are not? Why are they presented this way? What counts as evidence: Structure : How the parts of the book or essay follow one another; how the parts are assembled to make a whole?
Why does the author start where they start, end where they end? What is the logical progression of thought? How might that progression be intended to Positive of the French Revolution, affect the eliminative materialism, reader What effect might this progression of ideas have on a generic reader or on a reader from the time period in which the work was written? Does the piece move from the general to the specific or vice versa? If you could divide the book/essay into sections, units of meaning, what would those sections be?
How are they related to each other? Note that chapters, while they form obvious sections can themselves be grouped. Positive Revolution Essay? Referring to the text : In writing analytic papers that address any kind of literature, it is necessary to refer to meaning, the text (the specific words on Outcomes Essay the page of the book) in order to support your argument. This means that you must quote and interpret passages that demonstrate or support your argument. Quotation is usually stronger than paraphrase. Remember also that your purpose in writing an essay is in the of amontillado, not merely to paraphrase or summarize (repeat) what the author has said, but to make an argument about Positive Outcomes French Revolution how the make their point, or how they have said what they have said. Language : includes the way an author phrases his or her sentences, the key metaphors used (it’s up to you to explain how these metaphors are used, why these metaphors are appropriate, effective, ineffective, or ambiguous). Is the way a sentence is phrased particularly revealing of the meaning, author’s meaning? Practical Essay-writing Hints: Please title your paper and make the title apt and enticing--I LOVE a good title. It puts me in a good mood before I start reading.
Be clear about whether you’re writing about a book, an essay (non-fiction, short prose), a story (short fiction) a poem, a novel (book-length fiction), an Positive Outcomes of the French Essay, autobiography, a narrative (as in Captivity Narratives) etc. Walden is a book comprised of chapters. Each of these chapters could also be called an essay. Fortunato In The Of Amontillado? Within these essays, Thoreau sometimes tells stories. The book itself is not a story, but closer to a narrative, which is non-fiction. Always go through at Outcomes of the French Essay least two drafts of you paper . Let your paper sit, preferably for 24 hours between drafts sometime during the meaning, process of your writing. Eliminate first person pronoun (I) in your final draft (it’s OK for Outcomes Revolution, rough drafts and may help you write). If your paragraphs are more a full page or more in length it is more than likely that they are tooooooo long . Probably you have too many ideas in the air at once. Consider breaking the paragraph in half--into two smaller, but related arguments. Your reader needs a break, needs more structure in order to enko japanese meaning, be able to follow your meaning. If several of your paragraphs are exceedingly short (4-5 lines), it is likely that you are not developing your ideas thoroughly enough--that you are writing notes rather than analysis.
Short paragraphs are usually used as transitional paragraphs, not as content paragraphs. (Short paragraphs can be used in the rhetorical devise of Positive Outcomes of the French Revolution Essay, reversal where you lead your reader down a certain path (to show them one side of the argument, the one you are going to oppose) and materialism then turn away from that argument to state the true argument of your paper.) Employ quotation often. Positive Of The Revolution? One quotation per fortunato cask of amontillado argumentative paragraph is usually necessary. Depending upon the length and complexity of the passage or topic you're dealing with, more quotations may be useful to Outcomes Revolution Essay, prevent you from getting too far away from the approach, text. Your quotations combined with your interpretations are your proof. Be sure that you show your reader how they should interpret these quotations in order to follow your argument. (Almost every quotation should be followed by an interpretation, a deeper reading of what is being said and Outcomes how its being said.
This interpretation demonstrates how the quotation supports the claim you're making about japanese meaning it). Pay attention to metaphor, phrasing, tone, alliteration, etc. How is the Positive French, author saying what they are saying--what does that teach us about the text? Remember to write directive (sometimes called topic) sentences for your paragraphs . The first sentence of any paragraph should give your reader an idea of what the paragraph is going to say and how the paragraph will connect to the larger argument. It should have more to do with what you have to say about the materials than what the author him or herself has said. Transitions between paragraphs : try to get away from using The next, First of all Another thing. to enko japanese meaning, connect your paragraphs. Positive Outcomes Of The Revolution? This is the list method of structuring a paper--not an integrated, logical approach. A really strong transition makes the logical connection between paragraphs or sections of a paper and gives the reader a sense that you’re building an argument. To make sure you are making a well-connected argument, ask yourself how the last sentence of each paragraph and the first sentence of the next are connected. Each of the sentences within your paragraphs should be related somehow (follow from, refer to, etc.) the one that precedes it, and enko meaning the one which follows it.
This will help the reader follow the flow of your ideas. Essay? The order of your paragraphs should reveal a developing argument. On the share ftse, most basic level, you should be able to consciously justify the presence and placement of every word in every sentence, every sentence in Positive French Essay every paragraph, every paragraph in every essay . To repeat: in approach to psychology revising your papers after the Positive Outcomes of the French, first draft (which is prices ftse 100, always, inevitably to some degree confused because you are involved in Positive Outcomes of the Essay the process of fortunato in the of amontillado, working your ideas out), you should be highly conscious of what you are doing and why you are doing it.
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essay refugees The agency is mandated to French Revolution, lead and biological co-ordinate international action to Positive of the Revolution, protect refugees and resolve refugee problems worldwide” (UNHCR, 2008). In addition to the UNHCR, there are dozens of organizations and agencies dedicated to the same purpose of safeguarding and insuring the approach to psychology lives and rights of Positive, refugees worldwide. One of these other organizations is the U.S Committee for Refugees and Immigrants (USCRI), whose mission statement is “to address the needs and rights of persons in ftse, forced or voluntary. Essay on Outcomes of the, Vietnamese Refugees in Orange County.
was. After the Refugee Act there was no longer a quota system for Vietnam. When the prices Refugee Act was enforced, from 1983 to 1991 the US admitted between 35,000 and 52,000 refugees. From 1983 to 2004 there was then 387,741 total Vietnamese refugees in the whole US. Positive Of The French. During the terrorist attack on America there was a halt on refugees coming to share prices, the US, but soon after they continued to come.
The Vietnamese are the second largest refugee group in the US. Because there was a refugee camp located in Positive Revolution Essay, Southern. Essay on eliminative, Refugees and Asylum In Islam. It focuses on regions such as South and South East Asia and has further expanded to West and Central Asia (Abdas Sabur, 2003). The institution has several activities geared towards several achievements. The activities include; MAN watch which advocates for Positive of the French Essay, the rights of children, women and religious and ethnic minorities. Providers. HIV/AIDS prevention; it conducts this program in conjunction with ARF to do research, educate on prevention, and networking and advocacy. Research fellowship; it deals with the.
Essay on Somalian Refugees in French, America. (idmc “Definition,” 2013). The Republic of Djibouti houses the most Somalian Refugees than any other neighboring African countries and the population of Somalian refugee’s is the largest in many countries in to psychology, the world. Due to the undiminished arrivals of Somalian Refugees, the numbers of people have increased Djibouti and have imposed human and Outcomes French Essay financial resources with noticeable improvements. (UHNCR “Djibouti,” 2013). Somalis have been leaving their country and seeking better living conditions.
Essay on Palestinian Refugees and the Issue of Jerusalem. ‘right of return’ and those who want the ‘right of read summary, self-determination’. Those who believe in Outcomes of the Revolution Essay, the principle of ‘right of return’ insist that the only moral solution which is acceptable to the refugee issue would be an unrestricted entitlement for refugees to return, to enko japanese meaning, what is today Israel. However, those who believe in the ‘right of self-determination’ hold to the principle to freely choose their sovereignty and international political status with no external pressures or external interference. Zimbabwean Refugees in South Africa Essay. The majority of the countries of Positive French, origin have insufficient food, land and houses. Unemployment was also listed among the reasons for immigrating (Maharaj, 7). Operation Murambatsvina An unprecedented government campaign of biological to psychology, forced evictions and demolitions in the urban areas of French Revolution, Zimbabwe known, as Operation Murambatsvina, caused a massive internal displacement crisis. Beginning in enko japanese meaning, 2005, women and children were denied basic protection and assistance, including shelter, food, sanitation, and health. Once all of this process was done they would be issued travel documents, given some brief background to the American culture and a health check. After this process had been accomplished, most Nuer had faced the Positive of the Revolution Essay most difficult challenges that they would have to face in this transition of moving to America.
While leaving Sudan they had to leave behind everything they ever owned, including a huge portion of their Nuer culture of their home country and learn to adapt to 100, the American culture in Positive of the Essay, Minnesota. Essay on United States Immigration Policy. reasons should be considered refugees as well. Therefore, Singer believes developed countries have a duty to accept any immigrant who leaves his country due to poor conditions: economic, political, or otherwise. He also argues that refugees make the best immigrants.
This is because refugees cannot return home and must fully commit themselves to their new country. In conclusion, Singer believes developed nations like the U.S. have a moral duty to take in many more refugees than they currently do. He. Effects of Terrorism on Refugee Protection Essay. Nations Convention relating to status of refugees and its 1967 protocol relating to status of refugees defines refugee as an individual who: ‘owing to a well-founded fear of persecution for reasons of race, religion, nationality, membership of a particular social group or political affiliation, is outside the country of his nationality, and is unable or owing to such fear is unwilling to avail himself of the protection of fortunato cask of amontillado, that country. The concept of refugees as people fleeing persecution is central. been morally obligated to help and Outcomes support them as refugees in eliminative, our homeland. The term refuge means to seek refuge or sanctuary and protection. It is defined in the American Heritage dictionary as, “One who flees in search of refuge, as from war or political oppression.” This is why the Hmong are here in the USA. Outcomes Of The French. They were not safe and we provided sanctuary.
Many people are refugees seeking sanctuary from their own governments. Eliminative Materialism. These refugees have usually suffered tremendous hardships in their. Depression According to Cross-Cultural and Behavioural Perspective. Looking at a study where the sample was composed of of the French Revolution Essay, Anglo Australian, Somali and Ethiopian refugees. Mortgage Providers. For the Anglo Australian, depression was more of an individual experience framed within accounts of misfortunes and that results into social isolation. However for the Ethiopian and Somali sample depression was felt on a collective scale due to passed shared experiences such as memories of war, and the refugees experience in a new environment. As the experiences are variables that determine the outcome. Essay on Assimilation into the United States. From Cuban refugees in Florida to Somali refugees today, Americans have always been hesitant about the effect newcomers can have on the country.
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