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Specificity, efficiency, and fidelity of PCR.

1993; Cold Spring Harbor Laboratory Press; Volume: 3; Issue: 3 Linguagem: Inglês

10.1101/gr.3.3.s18

1549-5469

S. Rita, William G. Thilly,

Identification and Quantification in Food

The efficacy of PCR is measured by its specificity, efficiency (i.e.yield), and fidelity.A highly specific PCR will generate one and only one amplification product that is the intended target sequence.More efficient amplification will generate more products with fewer cycles.A highly accurate (i.e., high-fidelity) PCR, will contain a negligible amount of DNA polymerase-induced errors in its product.An ideal PCR would be the one with high specificity, yield, and fidelity.Studies indicate that each of these three parameters is influenced by numerous components of PCR, including the buffer conditions, the PCR cycling regime (i.e., temperature and duration of each step), and DNA polymerases.Unfortunately, adjusting conditions for maximum specificity may not be compatible with high yield; likewise, optimizing for the fidelity of PCR may result in reduced efficiency.Thus, when setting up a PCR, one should know which of the three parameters is the most important for its intended application and optimize PCR accordingly.For instance, for direct sequencing analysis of a homogenous population of ceils (either by sequencing or by RFLP), the yield and specificity of PCR is more important than the fidelity.On the other hand, for studies of individual DNA molecules, or rare mutants in a heterogeneous population, fidelity of PCR is vital.The purpose of current communication is to focus on the essential components of setting up an effective PCR, and discuss how each of these component may influence the specificity, efficiency, and fidelity of PCR. SETTING UP PCR TemplateVirtually all forms of DNA and RNA are suitable substrates for PCR.These include genomic (both eukaryotic and prokaryotic), plasmid, and phage DNA and previously amplified DNA, cDNA, and mRNA.Samples prepared via standard molecular methodologies (1) are sufficiently pure for PCR, and usually no extra purification steps are required.Shearing of genomic DNA during DNA extraction does not affect the efficiency of PCR (at least for the fragments that are less than -2 kb).In some cases, rare restriction enzyme digestion of genomic DNA before PCR is suggested to increase the yield.(2'3) In general, the efficiency of PCR is greater for smaller size template DNA (i.e., previously amplified fragment, plasmid, or phage DNA), than high molecular (i.e., undigested eukaryotic genomic) DNA.Typically, 0.1-1 pLg of mammalian genomic DNA is utilized per P e R . (1'3'4-6) Assuming that a haploid mammalian genome (3x109 bp) weighs -3 .4 x 10-az grams, 1 ~g of genomic DNA corresponds to -3 x 10 s copies of autosomal genes.For bacterial genomic DNA or a plasmid DNA that represent much less complex genome, picogram (10 -12 grams) to nanogram (10 -9 grams) quantities are used per reaction.(1'3) Previously amplified DNA fragments have also been utilized as PCR templates.In general, gel purification of the amplified fragment is recommended before the second round of PCR.Purification of the amplified product is highly recommended if the initial PCR generated a number of unspecific bands or if a different set of primers (i.e., internal primers) is to be utilized for the subsequent PCR.On the other hand, if the amplification reaction contains only the intended target product, and the purpose of the subsequent PCR is simply to increase the overall yield utilizing the same set of primers, no further purification is required.One could simply take out a small aliquot of the original PCR mixture and subject it to a second round of PCR.In addition to the purified form of DNA, PCR from cells has also been demonstrated.In this laboratory, direct amplification of hprt exon 3 fragment from 1 • 10 s human cells (following proteinase treatment to open up the cells) had been carried out routinely (P.Keohavong, unpubl.).