PCR Protocols: Current Methods and Applications

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Standardization of diagnostic PCR for the detection of foodborne pathogens. Int J Food Microbiol. Molecular and nanotechnologic approaches to etiologic diagnosis of infectious syndromes. Mol Diagn Ther. DNA amplification techniques in pharmacogenomics. Hepatitis B virus genotyping: current methods and clinical implications. Int J Infect Dis. A one-step real-time multiplex PCR for screening Y-chromosomal microdeletions without downstream amplicon size analysis. Prenatal diagnosis of hemoglobin disorders: present and future strategies. Clin Biochem. Systematic comparison of microarray profiling, real-time PCR, and next-generation sequencing technologies for measuring differential microRNA expression.

Benes V, Castoldi M. Hughes A. Methods Mol Biol. Jozefczuk J, Adjaye J. Quantitative real-time PCR-based analysis of gene expression. Twenty-five years of quantitative PCR for gene expression analysis. Automated DNA sequencing methods involving polymerase chain reaction. Clin Chem. Ansorge W. Next-generation DNA sequencing techniques. N Biotechnol. Foster A, Laurin N. Investig Genet. Morling N.

PCR in forensic genetics. Biochem Soc Trans. Decorte R, Cassiman J. Forensic medicine and the polymerase chain reaction technique. J Med Genet. Development of a locus PCR multiplex system for paternity testing. Int J Legal Med. J Struct Biol. Nelson M, Fitch D. Overlap extension PCR: an efficient method for transgene construction. Somatic APP gene recombination in Alzheimer's disease and normal neurons.

PAC, an evolutionarily conserved membrane protein, is a proton-activated chloride channel. L1 drives IFN in senescent cells and promotes age-associated inflammation. Nucleic Acids Res. Human placenta has no microbiome but can contain potential pathogens. Performance evaluation of thermal cyclers for PCR in a rapid cycling condition. The LightCycler: a microvolume multisample fluorimeter with rapid temperature control. Anal Chem. Centrifugal microfluidic system for primary amplification and secondary real-time PCR.

Lab Chip. Huhmer A, Landers J. Noncontact infrared-mediated thermocycling for effective polymerase chain reaction amplification of DNA in nanoliter volumes. A disposable, self-contained PCR chip. Plug-and-play, infrared, laser-mediated PCR in a microfluidic chip. Biomed Microdevices. Rozen S, Skaletsky H.

Primer3 on the WWW for general users and for biologist programmers. VizPrimer: a web server for visualized PCR primer design based on known gene structure.

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Thornton B, Basu C. Biochem Mol Biol Educ. PCR Methods Appl. Anal Biochem. Eischeid A. BMC Res Notes. This unit describes a method for amplifying DNA enzymatically by the polymerase chain reaction PCR , including procedures to quickly determine conditions for successful amplification of the sequence and primer sets of interest, and to optimize for specificity, sensitivity, and yield.

Once assembled, the mixture is cycled many times usually 30 through temperatures that permit denaturation, annealing, and synthesis to exponentially amplify a product of specific size and sequence. The PCR products are then displayed on an appropriate gel and examined for yield and specificity. Recommended optimization conditions are included. Many important variables can influence the outcome of PCR.

Careful titration of the MgCl 2 concentration is critical. The first stage steps to determines the optimal MgCl 2 concentration and screens several enhancing additives. NOTE : Use only molecular biology—grade water i. Enhancer agents optional; see recipe. Prepare four reaction master mixes according to the recipes given in Table 1. DMSO d d Substitute with other enhancer agents see recipe in 1 as available. Glycerol d d Substitute with other enhancer agents see recipe in 1 as available. PMPE d d Substitute with other enhancer agents see recipe in 1 as available. Substitute with other enhancer agents see recipe in 1 as available.

Enhancing agents probably work by different mechanisms, such as protecting enzyme activity and decreasing nonspecific primer binding. However, their effects cannot be readily predicted—what improves amplification efficiency for one primer pair may decrease the amplification efficiency for another. Thus it is best to check a panel of enhancers during development of a new assay. Similarly, aliquot mixes II through IV into appropriately labeled tubes.

Once the MgCl 2 has been added, do not allow the samples to cool below the optimum annealing temperature prior to performing PCR. Alternatives to mineral oil include silicone oil and paraffin beads. Additionally, certain cyclers feature heated lids that are designed to obviate the need for an oil overlay.

Using the following guidelines, program the automated thermal cycler according to the manufacturer's instructions. Cycling parameters are dependent upon the sequence and length of the template DNA, the sequence and percent complementarity of the primers, and the ramp times of the thermal cycler used. Thoughtful primer design will reduce potential problems see 2. Denaturation, annealing, and extension are each quite rapid at the optimal temperatures. The time it takes to achieve the desired temperature inside the reaction tube i. Thus, ramp time is a crucial cycling parameter.

Manufacturers of the various thermal cyclers on the market provide ramp time specifications for their instruments. The optimal extension time also depends on the length of the target sequence. The number of cycles depends on both the efficiency of the reaction and the amount of template DNA in the reaction.

With more template, fewer cycles may suffice. With much less template, further optimization is recommended rather than increasing the cycle number. Greater cycle numbers e. Many investigators lengthen the time for the last extension step—to 7 min, for example—to try to ensure that all the PCR products are full length.


PCR Protocols: Current Methods and Applications: 15 (Methods in Molecular Biology)

These guidelines are appropriate for most commercially available thermal cyclers. For rapid cyclers, consult the manufacturers' protocols. Stain with ethidium bromide. SeaKem increases the mechanical strength of the gel without decreasing resolution. Examine the stained gel to determine which condition resulted in the greatest amount of product.

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Minor, nonspecific products may be present even under optimal conditions. To ensure that the major product is the correct one, digest an aliquot of the reaction with a restriction endonuclease known to cut within the PCR product. Check buffer compatibility for the restriction endonuclease of choice. Electrophorese the digestion product on a gel to verify that the resulting fragments have the expected sizes.

With appropriately stringent hybridization and washing conditions, only the correct product and possibly some minor related products should hybridize. These optional steps optimize initial hybridization and may improve efficiency and yield.

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  • For an optimal reaction, polymerization during the initial denaturation and annealing steps should be prevented. Taq DNA polymerase activity can be inhibited by temperature reaction B , physical separation reaction C , or reversible antibody binding reaction D.

    PCR without hot start is performed for comparison reaction A. Prepare four reaction mixtures using the optimal MgCl 2 concentration and additive requirement determined in step. Prepare the mixes according to the recipes in Table 2.

    PCR protocols : current methods and applications

    Use the following variations for addition of Taq polymerase. Prepare reactions A and C at room temperature. For reaction D, combine 1. To ensure that the reaction does not plateau and thereby obfuscate the results, use the smallest amount of template DNA necessary for visualization of the PCR product by ethidium bromide staining. Use the results from step to decide how much template to use.

    If only a faint signal is apparent, use undiluted sample. Use undiluted or diluted template DNA based on results obtained in step. It is most convenient to use the automated thermal cycler for this step and then initiate the cycling program directly. Cool the reactions to the appropriate annealing temperature as determined in step. Add 0. Time is also an important factor in this step.

    If the temperature drops below the annealing temperature and is allowed to remain low, nonspecific annealing will occur. Taq DNA polymerase retains some activity even at room temperature. Begin amplification of all four reactions at once, using the same cycling parameters as before. Analyze the PCR products on an agarose gel and evaluate the results as in steps and 6. If desired, add Ficoll to a final concentration of 0.

    Adding Ficoll and tartrazine dye to the reaction mix precludes the need for a gel loading buffer and permits direct application of PCR products to agarose or acrylamide gels.

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    Other dyes, such as bromphenol blue and xylene cyanol, do inhibit PCR. Tartrazine is a yellow dye and is not as easily visualized as other dyes; this may make gel loading more difficult. Use deionized, distilled water in all recipes and protocol steps. For a discussion of how to select enhancer agents, see 2. The theoretical basis of the polymerase chain reaction PCR; see chapter introduction was probably first described in a paper by Kleppe et al. The initial procedure entailed adding a fresh aliquot of the Klenow fragment of E.

    Taq DNA polymerase also permitted the use of higher temperatures for annealing and extension, which improved the stringency of primer—template hybridization and thus the specificity of the products. This also served to increase the yield of the desired product. These and other parameters can be extremely important, as every element of PCR can affect the outcome see 2.

    There are several PCR optimization kits and proprietary enhancers on the market Table 3. Optimization kits generally provide a panel of buffers in which the pH, buffer, nonionic detergents, and addition of NH 4 2 SO 4 are varied, MgCl 2 may be added at several concentrations, and enhancers e.

    The protocol presented here is aimed at keeping the costs low and the options broad. PCR SuperMix—1. Determining the optimum MgCl 2 concentration, which can vary even for different primers from the same region of a given template Saiki, , can have an enormous influence on PCR success. In this protocol three concentrations are tested—1. Enhancers tend to broaden the MgCl 2 optimal range, contributing to the success of the PCR at one of these concentrations. For applications that amplify rare templates, reagent purity is the most important parameter, and avoiding contamination at every step is critical.

    Even tiny amounts of chemical left after treatment of water by autoclaving are enough to ruin a PCR. This is the factor that is least predictable and most difficult to troubleshoot. Simply put, some primers just do not work. To maximize the probability that a given primer pair will work, pay attention to the following parameters.

    General considerations. An optimal primer set should hybridize efficiently to the sequence of interest with negligible hybridization to other sequences present in the sample. If there are reasonable amounts of template available, hybridization specificity can be tested by performing oligonucleotide hybridization as described in UNIT Unavailable.

    The distance between the primers is rather flexible, ranging up to 10 kb. Small distances between primers, however, lessen the ability to obtain much sequence information or to reamplify with nested internal oligonucleotides, should that be necessary. Design primers to allow demonstration of the specificity of the PCR product. Be sure that there are diagnostic restriction endonuclease sites between the primers or that an oligonucleotide can detect the PCR product specifically by hybridization.

    Several computer programs can assist in primer design see Internet Resources at end of unit. Given the abundance of primers relative to template, this can preclude template priming. Computer primer design is not foolproof. If possible, start with a primer or primer set known to efficiently prime extensions.

    In addition, manufacturers' Web sites offer technical help with primer design. Complementarity to template. For many applications, primers are designed to be exactly complementary to the template. It is best to have mismatches e. The use of degenerate oligonucleotide primers to clone genes where only protein sequence is available, or to fish out gene homologs in other species, has sometimes been successful, but it has also failed an untold and unpublished number of times. When the reaction works it can be extremely valuable, but it can also generate seemingly specific products that require much labor to identify and yield no useful information.

    Caveat emptor. Primer length. A primer should be 20 to 30 bases in length. It is unlikely that longer primers will help increase specificity significantly. Primer sequence. Design primers with a GC content similar to that of the template. Avoid primers with unusual sequence distributions, such as stretches of polypurines or polypyrimidines, as their secondary structure can be disastrous. It is worthwhile to check for potential secondary structure using one of the appropriate computer programs that are available.

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    The ensuing product can compete very effectively against the PCR product of interest. Should they occur, optimizing the MgCl 2 concentration may minimize their abundance relative to that of the product of interest. The two main concerns regarding template are purity and amount.

    Reverse transcription PCR - RT PCR procedure
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