The polymerase chain reaction

10 Apr

5 The polymerase chain reaction
In 1985, the year of the first DNA ‘fingerprint’, a new method – the polymerase chain reaction (PCR) was reported [1-3]. The PCR can amplify a specific region of DNA and it has revolutionized all areas of molecular biology, including forensic genetics. The technique allows extremely small quantities of DNA to be amplified. Under optimal conditions, DNA can be amplified from a single cell [4, 5]. The increased sensitivity of DNA profiling using PCR has had a dramatic effect on the types of forensic sample that can be used and it is now possible to analyse trace evidence and highly degraded samples successfully – albeit with less than 100% success.

The evolution of PCR-based profiling in forensic genetics
PCR technology was rapidly incorporated into forensic analysis. The first PCR based tool for forensic casework amplified the polymorphic HLA DQα locus (the α subunit of the DQ protein is part of the major histocompatibility complex) [6]. It was used for the first time in casework in 1988 to analyse the skeletal remains of a 3-year-old girl [7, 8]. The DQα system’s major drawback was that it had a limited power of discrimination. VNTRs were widely used in casework but required a relatively large amount of DNA. In an attempt to overcome this limitation, PCR technology was applied to the analysis of VNTR loci, and alleles between 5-10 kb could be faithfully amplified from fresh biological material [9]. However, it was of limited value for many forensic samples, which often contained small amounts of DNA that could be highly degraded. To overcome the problems caused by degradation, tandem repeats, called AMP-FLPs (amplified fragment length polymorphisms), that were smaller than 1 kb were selected for PCR based analysis [10-15]. However, as with VNTRs, their use was limited in forensic contexts because of the size of the larger alleles, which were difficult to analyse in degraded samples. By the early 1990s, a large number of short tandem repeats (STRs) had been characterized [16]. The STR loci were simpler and shorter than VNTRs and AMP-FLPs, and were more suitable for the analysis of biological samples recovered from crime scenes [17]. The STR markers were not individually as discriminating as the VNTR and AMP-FLPs but had a major advantage that several of them could be analysedtogetherinamultiplexreaction.Theshorttandemrepeatmarkershavebecome

An Introduction to Forensic Genetics  W. Goodwin, A. Linacre and S. Hadi
© 2007 John Wiley & Sons, Ltd

the genetic polymorphism of choice in forensic genetics and the PCR is a vital part of the analytical process.

DNA replication – the basis of the PCR
The PCR takes advantage of the enzymatic processes of DNA replication. During every cell cycle the entire DNA content of a cell is duplicated. This copying of DNA can be replicated outside of the cell in vitro to amplify specific regions of DNA.

The components of PCR
A PCR has the following components: template DNA, at least two primers, a ther-mostable DNA polymerase such as Taq polymerase, magnesium chloride, deoxynu- cleotide triphosphates and a buffer.

Template DNA
The amount of DNA added to a PCR depends on the sensitivity of the reaction: for most forensic purposes the PCR is highly optimized so that it will work with low levels of template. Most commercial kits require between 0.5 and 2.5 ng of extracted DNA for optimum results. This represents between 166 and 833 copies of the haploid human genome – one copy of the human genome contains approximately 3 pg of DNA. Most forensic profiling can be carried out successfully with fewer templates – even below 100 pg or 33 copies of the genome; however, the interpretation of profiles can become more complex as the amount of template DNA is reduced.

Taq DNA polymerase
The first PCRs were carried out using a DNA polymerase that was isolated from E. coli; in each cycle of the PCR the enzyme was inactivated by the high tempera- tures in the denaturation phase and fresh enzyme had to be added [1-3]. Fortunately this is no longer necessary. Scientists were able to isolate the DNA polymerases from the thermophilic bacteria, Thermus aquaticus [18], which was discovered in the 1960s in the hot springs of Yellowstone National Park, USA. The Taq polymerase enzyme can tolerate the high temperatures that are involved in the PCR and works optimally at 72-80 ◦C [19]. Using the thermostable enzyme greatly simplifies the PCR procedure and also increases the specificity, sensitivity and yield of the reaction [19]. The Taq polymerase enzyme exhibits significant activity at room temperature that can lead to the creation of non-specific PCR products; adding the enzyme to a pre-heated ‘hot start’ reaction reduces the non-specific binding and again improves the specificity and yield of a PCR [20]. Modifications to the commonly used Taq polymerase led to the development of the AmpliTaq Gold®  polymerase (Applied Biosystems). The enzyme

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