However, if fidelity is more important an enzyme such as Pfu may be a better choice. Several manufactures have an array of specific DNA polymerases designed for specialized needs. Take a look at the reaction conditions and characteristics of the desired amplicon, and then match the PCR experiment with the appropriate DNA polymerase.
Most manufactures have tables that aid DNA polymerase selection by listing characteristics such as fidelity, yield, speed, optimal target lengths, and whether it is useful for G-C rich amplification or hot start PCR. Optimal target molecules are between 10 4 to 10 7 molecules and may be calculated as was described in the notes above. Additive reagents may yield results when all else fails.
Understanding the reagents and what they are used for is critical in determining which reagents may be most effective in the acquisition of the desired PCR product. Adding reagents to the reaction is complicated by the fact that manipulation of one reagent may impact the usable concentration of another reagent. In addition to the reagents listed below, proprietary commercially available additives are available from many biotechnology companies.
Formamide final reaction concentration of 1. Formamide also has been shown to be an enhancer for G-C rich templates. As the amplicon or template DNA is denatured, it will often form secondary structures such as hairpin loops. Betaine final reaction concentration of 0. Non ionic detergents function to suppress secondary structure formation and help stabilize the DNA polymerase.
Non ionic detergents such as Triton X, Tween 20, or NP may be used at reaction concentrations of 0. The presence of non ionic detergents decreases PCR stringency, potentially leading to spurious product formation. However, their use will also neutralize the inhibitory affects of SDS, an occasional contaminant of DNA extraction protocols. Hot start PCR is a versatile modification in which the initial denaturation time is increased dramatically Table 4.
This modification can be incorporated with or without other modifications to cycling conditions. Moreover, it is often used in conjunction with additives for temperamental amplicon formation. In fact, hot start PCR is increasingly included as a regular aspect of general cycling conditions. Hot start has been demonstrated to increase amplicon yield, while increasing the specificity and fidelity of the reaction.
The rationale behind hot start PCR is to eliminate primer-dimer and non-specific priming that may result as a consequence of setting up the reaction below the T m. In general, the DNA polymerase is withheld from the reaction during the initial, elongated, denaturing time. Although other components of the reaction are sometimes omitted instead of the DNA polymerase, here we will focus on the DNA polymerase.
There are several methods which allow the DNA polymerase to remain inactive or physically separated until the initial denaturation period has completed, including the use of a solid wax barrier, anti-DNA polymerase antibodies, and accessory proteins. Alternatively, the DNA polymerase may simply be added to the reaction after the initial denaturation cycle is complete. The concept is to design two phases of cycling conditions Table 5. The first phase employs successively lower annealing temperatures every second cycle traditionally 1.
The function of the first phase should alleviate mispriming, conferring a 4-fold advantage to the correct product.
Thus, after 10 cycles, a fold advantage would yield copies of the correct product over any spurious priming. This would allow the correct product a fold advantage over false priming products. The concept takes into account a relatively new feature associated with modern thermal cyclers, which allows adjustment of the ramp speed as well as the cooling rate.
The ramp speed is lowered to 2. Nested PCR is a powerful tool used to eliminate spurious products. The use of nested primers is particularly helpful when there are several paralogous genes in a single genome or when there is low copy number of a target sequence within a heterogeneous population of orthologous sequences.
The basic procedure involves two sets of primers that amplify a single region of DNA. The outer primers straddle the segment of interest and are used to generate PCR products that are often non-specific in 20 to 30 cycles.
Other PCR protocols are more specialized and go beyond the scope of this paper. The results incorporate several troubleshooting strategies to demonstrate the effect of various reagents and conditions on the reaction. Genes from the budding yeast Saccharomyces cerevisiae and from an uncharacterized Mycobacteriophage were amplified in these experiments.
The standard 3-step PCR protocol outlined in Table 2 was employed for all three experiments described below. The working stocks were prepared as follows. Since the S. This phage DNA is about 67 Kb. Thus, 1 ng contains 2. The working stocks were then used to generate the Master Mix solutions outlined in Table 7.
Experiments varied cycling conditions as described below. No MgCl 2 was present in the original PCR buffer and had to be supplemented at the concentrations indicated with a range tested from 0. The recommended concentration provided by the manufacturer was 1.
Perhaps surprisingly, the necessary concentration needed for product formation in this experiment exceeded this amount. A different DNA template was used for the experiment presented in Figure 3b. As shown in Figure 3b , amplification of the desired PCR product requires at least 2. Notice that in the experiments presented in Figures 3A and 3B , a discrete band was obtained using the cycling conditions thought to be optimal based on primer annealing temperatures.
For the third experiment presented in Figure 3c , three changes were made to the cycling conditions used to amplify the yeast GAL3 gene. Second, the extension time was extended to 1 minute and 30 seconds. Third, the number of cycles was increased from 30 to 35 times. The purpose was to demonstrate the effects of sub-optimal amplification conditions i. As shown in Figure 3c , what was a discrete band in Figure 3a , becomes a smear of non-specific products under these sub-optimal cycling conditions.
These results also demonstrate that when both the cycling conditions are correctly designed and the reagents are at optimal concentrations, the PCR experiment produces a discreet amplicon corresponding to the expected size. The results show the importance of performing PCR experiments at a sufficiently high stringency e.
Moreover, the experiments indicate that changing one parameter can influence another parameter, thus affecting the reaction outcome. The Master Mix depicted in the above table is calculated for 11 reactions plus 2 extra reactions to accommodate pipette transfer loss ensuring there is enough to aliquot to each reaction tube. Table 7. Figure 1. Note that primers do not always anneal at the extreme ends and may form smaller loop structures.
Once the primers anneal to each other they will elongate to the primer ends. Figure 2. Ice bucket with reagents, pipettes, and racks required for a PCR. P pipette, 2. P pipette, 3. P pipette, 4. P pipette, 5. Figure 3. Lanes 9 - 11 are indicative of excessively stringent conditions with no product formed.
Figure 4. Sterile tubes used for PCR. Figure 5. Thermal cycler. Closed thermal cycler left image. Right image contains 0. PCR has become an indispensible tool in the biological science arsenal. PCR has altered the course of science allowing biologists to yield power over genomes, and make hybrid genes with novel functions, allowing specific and accurate clinical testing, gaining insights into genomes and diversity, as well as simply cloning genes for further biochemical analysis.
PCR application is limited only by the imagination of the scientist that wields its power. There are many books and papers that describe new specialized uses of PCR, and many more will be developed over the next generation of biological science. However, regardless of the anticipated approaches, the fundamental framework has remained the same. PCR, in all its grandeur, is an in vitro application to generate large quantities of a specific segment of DNA.
Designing a PCR experiment requires thought and patience. The results shown in Figure 3 exemplify one of the major challenges when designing an optimization strategy for PCR. That is, as one parameter of PCR is changed, it may impact another. An attempt to resolve the smear might involve setting up PCR conditions with reactions containing 2. However, as seen in Figure 3a, this would not yield any product. Consequently, it is advisable to titrate reagents, rather than adding one concentration to a single reaction, when troubleshooting spurious results.
If all else fails, redesign the primers and try, try again. I would also like to thanks Giancarlo Costaguta and Gregory S. I would also like to thank Bhairav Shah for taking pictures of the lab equipment and reagents used to make figures 2 - 4.
National Center for Biotechnology Information , U. J Vis Exp. Published online May Todd C. Lorenz 1. Author information Copyright and License information Disclaimer. Correspondence to: Todd C. Lorenz at ude. This article has been cited by other articles in PMC. Abstract In the biological sciences there have been technological advances that catapult the discipline into golden ages of discovery. Keywords: Basic Protocols, Issue 63, PCR, optimization, primer design, melting temperature, T m , troubleshooting, additives, enhancers, template DNA quantification, thermal cycler, molecular biology, genetics.
Download video file. Protocol 1. Designing Primers Designing appropriate primers is essential to the successful outcome of a PCR experiment. Below is a list of characteristics that should be considered when designing primers. Primer length should be nucleotide residues bases. Notes: There are many computer programs designed to aid in designing primer pairs.
Organize laboratory equipment on the workbench. Setting up a Reaction Mixture Start by making a table of reagents that will be added to the reaction mixture see Table 1. Next, label PCR tube s with the ethanol-resistant marker. Notes: When setting up multiple PCR experiments, it is advantageous to assemble a mixture of reagents common to all reactions i. Manipulating PCR Reagents Understanding the function of reagents used on conventional PCR is critical when first deciding how best to alter reaction conditions to obtain the desired product.
Additive Reagents Additive reagents may yield results when all else fails. Open in a separate window. Discussion PCR has become an indispensible tool in the biological science arsenal. Disclosures I have nothing to disclose. Simple sensitive, and specific detection of human immunodeficiency virus type 1 in clinical specimens by polymerase chain reaction with nested primers.
Uniform amplification of a mixture of deoxyribonucleic acids with varying GC content. Genome Res. PCR buffer optimization with uniform temperature regimen to facilitate automation.
PCR Methods Appl. Stability of ribonucleic acid double-stranded helices. Predicting DNA duplex stability from the base sequence. Prevention of pre-PCR mis-priming and primer dimerization improves low-copy-number amplifications. Nucleic Acids Res. Diagnostics for the clinical laboration. Humana Press; Maximizing sensitivity and specificity of PCR by pre-amplification heating. General concepts for PCR primer design. Recent advances in the polymerase chain reaction. PCR Protocols: A guide to methods and applications.
San Diego: Academic Press, Inc; Methods in Molecular Biology. Studies on polynucleotides. The polymerase chain reaction. Protein Sci. Relief of amplification inhibition in PCR with bovine serum albumin or T4 gene 32 protein.
Identification of human immunodeficiency virus sequences by using in vitro enzymatic amplification and oligomer cleavage detection. The unusual origin of the polymerase chain reaction. Methylation-Specific PCR. Cloning PCR Products. Autosticky PCR. Tamara S. Gritsun, Michael V. Mikhailov, Ernest A. Page 1 Navigate to page number of 3. Here the researcher will find readily reproducible methods for all the major aspects of PCR use, including PCR optimization, computer programs for PCR primer design and analysis, and novel variations for cloning genes of special characteristics or origin, with emphasis on long-distance PCR and GC-rich template amplification.
Also included are both conventional and novel enzyme-free and restriction site-free procedures to clone PCR products into a range of vectors, as well as state-of-the-art protocols to facilitate DNA mutagenesis and recombination and to clone the challenging uncharacterized DNA flanking a known DNA fragment.
Powerful applications of PCR in library construction and sublibrary generation and screening are also presented. Editors and affiliations. Bing-Yuan Chen 1 Harry W. Janes 1 1. Reviews "This hardcover book is packed full of detailed protocols and methodological articles on various aspects of PCR. The fact that the book was taken out of my hands by a colleague, to read up on touch-down PCR, as soon as I received it for review, attest to the book's usefulness.
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