29 YEARS ON, A NOBEL PRIZE, AND A DIAGNOSTIC DREAM: THE USES OF THE POLYMERASE CHAIN REACTION IN MODERN BIOLOGY

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1 The West London Medical Journal 2012 Vol 4 No 1 pp YEARS ON, A NOBEL PRIZE, AND A DIAGNOSTIC DREAM: THE USES OF THE POLYMERASE CHAIN REACTION IN MODERN BIOLOGY Jason Seewoodhary Specialist Registrar in Diabetes Mellitus & Endocrinology / General Internal Medicine Department of Diabetes and Endocrinology University Hospital of Wales Heath Park Cardiff CF14 4XN United Kingdom. seewoodharyj@hotmail.com ABSTRACT This review will critically consider the uses of the polymerase chain reaction (PCR) in modern biology since its pioneering advent in PCR is an indispensible tool for use in: DNA cloning for sequencing; DNA-based phylogeny; forensic applications; and diagnostic clinical medicine, which involves diagnosing hereditary diseases, tumours and infectious diseases. The process of PCR will be outlined followed by a discussion on these uses, which will consider the limitations of PCR and how these have been overcome by the development of variations in standard PCR protocols. Keywords: PCR; DNA Cloning; Sequencing; Diagnostics; Forensics INTRODUCTION This review will critically consider the uses of the polymerase chain reaction (PCR) in modern biology since its pioneering advent in PCR is an indispensible tool for use in: DNA cloning for sequencing; DNA-based phylogeny; forensic applications; and diagnostic clinical medicine, which involves diagnosing hereditary diseases, tumours and infectious diseases. The process of PCR will be outlined followed by a discussion on these uses, which will consider the limitations of PCR and how these have been overcome by the development of variations in standard PCR protocols. 7

2 THE WEST LONDON MEDICAL JOURNAL , 1 PCR is an in vitro enzymatic technique that exponentially amplifies a target DNA sequence. It is an automated method that relies of thermal cycling, which involves alternately heating and cooling the PCR sample to a defined series of temperature steps. The first step involves denaturating dsdna by heating the sample to 94-96ºC for 1 minute. This exposes the target sequence on ssdna. The second stage involves the annealing of primers, which occurs optimally at a lower temperature of 54ºC for 45 seconds. Ionic bonds form between the primer and template and it is on that primer-dna template that DNA polymerase begins adding nucleotides onto pre-existing 3 -OH groups thus copying the template during the extension phase. During extension the temperature is increased to 72 ºC for 2 minutes, which is the optimal temperature for DNA polymerase enzymes, such as Taq polymerase. This is a heat resistant enzyme that in the presence of dntp s and divalent cations such as Mg 2+, synthesizes new strands of DNA complementary to template DNA. Each PCR cycle doubles the number of DNA molecules; the DNA generated is used as a template for replication in the following cycle, which amplifies target DNA by several orders of magnitude [1]. PCR has played a major role in DNA cloning for sequencing. The main advantages of PCR as a cloning method are its rapidity, sensitivity and robustness. DNA cloning by PCR is performed in a few hours, which compares favorably with cell-based DNA cloning, which may take weeks and is labour intensive. PCR is exquisitely sensitive, being able to amplify sequences from minute amounts of target DNA from single cells. However, the high sensitivity of PCR is offset by the need to take extreme care to avoid contamination of the sample by external DNA. The robustness of PCR allows DNA that is degraded or embedded in a medium to be amplified, which would be difficult with conventional cloning techniques. This lends PCR to being suitable for molecular anthropology and paleontology studies, where DNA recovered from archaeological remains maybe damaged and it is on this principle that PCR has revolutionized phylogenetics. Prior to the advent of PCR, phylogenetic relationships were reconstructed by looking at anatomical phenotypic characteristics. PCR has advanced molecular phylogenetics by generating molecular sequencing data, which has helped to determine the rates and patterns of diversification and changes in DNA and proteins. Such data has been used to reconstruct the evolutionary history of genes and organisms [2]. In anthropology PCR has been used to understand ancient human migration patterns. The role PCR has in DNA cloning for sequencing permits rapid genotyping of polymorphic markers. PCR can type Restriction Site Polymorphisms (RSPs) by designing primers using sequences that flank the polymorphic restriction site to produce a short product. Digestion of the PCR product with restriction enzymes and size fractionation can result in simple typing of the alleles. This provides an alternative to the conventional Restriction Fragment Length Polymorphism (RFLP) assays, which are time 8

3 29 YEARS ON, A NOBEL PRIZE, AND A DIAGNOSTIC DREAM: THE USES OF THE POLYMERASE CHAIN REACTION IN MODERN BIOLOGY consuming and laborious. The role PCR has in DNA cloning for sequencing also allows typing of Short Tandem Repeat Polymorphisms (STRPs) [3]. This impacts upon the role of PCR in forensic and diagnostic clinical applications. PCR has a major role in forensics due to the high information potential at DNA level especially with regards to highly polymorphic DNA segments, such as minisatellites or Variable Number Tandem Repeats (VNTR s), which can generate individual specific DNA fingerprints [4]. This compares favourably to the lower discrimination potential of electrophoretic methods and serological markers that were traditionally used to determine the origin of forensic material. PCR negates problems associated with DNA typing of forensic material, such as insufficient or degraded DNA. The high sensitivity of PCR means that large amounts of DNA are not needed for typing polymorphisms, which affords the advantage that repeat analysis is possible in cases where defence lawyers require confirmation of the results. This contrasts favourably to conventional DNA typing methods, which require ~50-500ng of high molecular weight DNA, meaning repeat analysis is not feasible. PCR has also made it possible to use other biological material to associate suspects with a crime, such as single hairs, epithelial cells from urine, buccal epithelial cells, and fingernails. Cigarette butts contain saliva, but only small amounts of DNA (10 to 100ng) can be extracted which is insufficient for Southern blot analyses. Minute quantities of saliva on stamps and envelopes can be a source of DNA to identify suspects involved in blackmail. The downside of using PCR in forensic biology is a consequence of its high sensitivity, which raises concerns about accidental contamination of DNA samples or PCR reactions, which was the crux of the defence case in recent the Stephen Lawrence murder trial. PCR is the most sensitive method for detecting and characterizing microorganisms in clinical specimens and is indicated when specific pathogens are difficult to culture in vitro such as Mycobacterium tuberculosis. The most frequently used targets for amplification of M. tuberculosis DNA is the insertion element IS6110, which is present in multiple copies and is specific for strains belonging to the M. tuberculosis complex [5]. This contributes to the higher sensitivity of PCR compared with that of single-copy targets. However, the diagnostic clinical uses of PCR are not without problems; false-positive results may occur due to contamination with amplicons from previous reactions and false-negative results may arise from the presence of inhibitors of the PCR. Examples of inhibitors from samples (blood, sputum, and pleural fluid) include haemoglobin and heparin, which may persist even after DNA purification. To control for this an internal control is needed, which would be able to detect inhibition. This also allows the PCR results to be quantified, providing useful clinical data for clinicians during follow-up of patients on anti-tuberculous treatment. PCR allows for rapid prenatal diagnosis and carrier testing of several hereditary disorders. Inherited variations in DNA can be detected with 9

4 THE WEST LONDON MEDICAL JOURNAL , 1 unamplified DNA or by using PCR to amplify DNA. Results from analysis of amplified DNA can be obtained within a day relative to weeks or even months when unamplified DNA is analyzed [6]. After PCR, mutations can be detected by several methods, which include: endonuclease digestion when a mutation affects an endonuclease recognition site; gel electrophoresis for detection of deletions; and hybridization to an oligonucleotide probe specific for a mutation. Gene sequencing of a PCR product can be used to rapidly identify a mutation. Furthermore, PCR can be applied to polymorphism analysis to provide a diagnosis by linkage analysis. Despite the widespread uses of PCR in modern biology, the technique is not without limitations. Taq polymerase lacks 3 to 5 exonuclease activity and thus cannot correct misincorporated nucleotides during PCR, which is associated with an error rate of ~ 1 in 10,000 bases incorporated. However, recombinant polymerases, such as Vent have been generated and other polymerases, such as Pfu and Pwo have been isolated that possess 3 to 5 exonuclease activity, affording higher fidelity. These enable more accurate PCR for a variety of applications such as sequencing [7]. Furthermore, whilst DNA polymerases are efficient in amplifying DNA segments up to ~2-5 kb, such efficiency is lost with larger segments due to enzyme activity loss and inaccuracies during longer PCRs. Whilst adding fresh DNA polymerase helps with enzyme activity lost due to the half life of the polymerase this does not help when accurate PCR is required. It is possible to amplify PCR products up to 20kb using slower heating cycles and special mixtures of polymerases [8]. These limitations can be offset by a number of variants and small modifications to the standard PCR protocol. This includes: Multiplex PCR, which permits the simultaneous analysis of multiple targets in a single sample; Quantitative PCR, which is used to measure the specific amount of DNA in a sample; Nested PCR, used to increase the specificity of DNA amplification; Reverse Transcription PCR for amplifying DNA from RNA; Real Time PCR, which permits the analysis of the products while the reaction is in progress; Inverse PCR; Solid Phase PCR; Methylation Specific PCR; Ligation mediated PCR; and Miniprimer PCR. In Conclusion, PCR has transformed biology laboratories and its limitations have been offset by varying the standard protocol. Its significance is evidenced by the award of the Nobel Prize in chemistry to its pioneers. PCR has book-marked its place in scientific discoveries and undoubtedly more uses will be unravelled in the future. REFERENCES 1. Mullis K, The unusual origin of the polymerase chain reaction. Scientific American 262 (1990): pp Witthuhn WC, Wingfield BD et al., PCR-based identification and 10

5 29 YEARS ON, A NOBEL PRIZE, AND A DIAGNOSTIC DREAM: THE USES OF THE POLYMERASE CHAIN REACTION IN MODERN BIOLOGY phylogeny of species of Ceratocystis sensu stricto. Mycological Research 103 (1999): pp Levitt RC, Molecular genetic methods for mapping disease genes. Am J Respir Crit Care Med 150 (1994): pp. S Jobbling MA, Gill P, Encoded evidence: DNA in forensic analysis. Nature Reviews Genetics 5 (2004): pp Almeda J, Garcia A et al., Clinical evaluation of an in-house IS6110 polymerase chain reaction for diagnosis of tuberculosis. Eur J Microbiol Infect Dis 19 (2000): pp Rommens JM, Januzzi MC et al., Identification of the cystic fibrosis gene: chromosome walking and jumping. Science 245 (1989): pp Fazlieva R, Spittle CS et al., Proofreading exonuclease activity of human DNA polymerase δ and its effects on lesion-bypass DNA synthesis. Nucleic Acids Res 37 (2009): pp Her C, Weinshilboum RM., Endonuclease-Mediated Long PCR and Its Application to Restriction Mapping. Current Issues Molec. Biol 1 (1999): pp