Polymerase Chain Reaction (PCR): Specialised Applications

Abstract

The polymerase chain reaction (PCR) is a technique that rapidly amplifies selected subsets of deoxyribonucleic acid (DNA) or complementary DNA (cDNA) from initially complex biological mixtures. The efficiency of amplification or sequence of the amplified material can then be examined for many purposes, including genotyping or the characterisation of new genes, gene expression patterns, mutations or polymorphisms. Quantitative‐PCR (QT‐PCR) has become practical and popular over the past decade with the generation of libraries of individual primer sets and arrayed sets of primers by commercial entities. QT‐PCR machines can assay up to 384 samples at once, permitting the complete analysis of expression of families of genes, such as signalling kinases or cytokines, in a single experiment. Rapid amplification of cDNA ends‐PCR has also become popular to identify microRNAs (ribonucleic acid). PCR is highly useful in structural biology to rapidly modify the boundaries of protein domains intended for crystallisation and in genomics for high‐throughput sequencing.

Key Concepts:

  • PCR can be conducted on vanishingly small amounts of material.

  • PCR can be used for rapid genotyping or sequencing.

  • PCR is useful for quantitative analysis of gene expression of entire gene families or signalling networks in arrayed assays.

  • PCR is frequently the method of choice for genetic engineering involving mutations, truncations or extensions of existing genes.

  • PCR is a preferred method for high‐thoughput library screens.

Keywords: RACE‐PCR; RT‐PCR; polymorphisms; genotyping

Figure 1.

Schematic representation of classic RACE. GSP1, gene‐specific primer 1; GSP‐RT, gene‐specific primer used for reverse transcription.

Figure 2.

Schematic representation of new RACE. This method differs from classic RACE in that the tag (an RNA oligo) is appended to the mRNA before reverse transcription. Accordingly, only cDNAs that are successfully extended all the way to the 5′ end of the mRNA encode the tag and thus become templates capable of being amplified by the RACE primers.

Figure 3.

Accumulation of PCR products during successive cycles of amplification. Amplification profiles are shown for two samples that differ eightfold in the starting quantity of template. Note that although the eightfold difference is easily discerned during the logarithmic phase of amplification for both samples (i.e. within the dotted box), examining the output at cycle 26 (arrow) or later would lead to the incorrect conclusion that the original samples had contained equal starting amounts of material, because all amplifications eventually cease at the same plateau.

Figure 4.

Schematic representation of PCR genotyping, when a new restriction site has been introduced in the mutant allele.

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References

Frohman MA, Dush MK and Martin GR (1988) Rapid production of full‐length cDNAs from rare transcripts by amplification using a single gene‐specific oligonucleotide primer. Proceedings of the National Academy of Sciences of the USA 85: 8998–9002.

Morrison TB, Weis JJ and Wittwer CT (1998) Quantification of low‐copy transcripts by continuous SYBR green I monitoring during amplification. BioTechniques 24: 954–962.

Schalasta G and Schmid M (1999) Ultrarapid and semiautomated real‐time PCR – a breakthrough in nucleic acid analysis. Clinical Laboratory 45: 661–663.

Scotto‐Lavino E, Du G and Frohman MA (2006) Amplification of 5′ end cDNA with ‘new RACE’. Nature Protocols 1: 3056–3061.

Shalem O, Sanjana NE, Hartenian E et al. (2014) Genome‐scale CRISPR‐Cas9 knockout screening in human cells. Science 343: 84–87.

Tyagi S and Kramer FR (1996) Molecular beacons–probes that fluoresce upon hybridization. Nature Biotechnology 14: 303–308.

Further Reading

Letts VA, Felix R, Biddlecome GH et al. (1998) The mouse stargazer gene encodes a neuronal Ca2+‐channel gamma subunit. Nature Genetics 19: 340–347.

Louis C, Madueno E, Modolell J et al. (1997) One‐hundred and five new potential Drosophila melanogaster genes revealed through STS analysis. Gene 195: 187–193.

Spelman R and Bovenhuis H (1998) Genetic response from marker assisted selection in an outbred population for differing marker bracket sizes and with two identified quantitative trait loci. Genetics 148: 1389–1396.

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How to Cite close
Frohman, Michael A(Sep 2014) Polymerase Chain Reaction (PCR): Specialised Applications. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0003152.pub3]