DNA Replication Methods: PCR and Whole Genome Replication
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DNA Replication Methods: PCR and Whole Genome Replication

Brief explanation of PCR, polymerase chain reaction, and WGA (Whole Genome Amplification).

PCR (Polymerase chain reaction) was invented in 1986 by Kary Mullis at Cetus Corporation in California, two years after Jeffreys' used RFLP and accidentally created DNA fingerprinting. The PCR technique affected many different areas of science including forensics where it eventually replaced Jeffreys RFLP system for typing DNA. The PCR technique is so essential to forensic and molecular biology that it is used in almost every procedure where it is necessary to increase the quantity of DNA for the purposes of testing it. Today, DNA profiles utilize Short Tandem Repeat (STR) loci. Like the SLP probes used in RFLP and the SNPs used for medical testing, STR have a low amount of allele variation, in this case about 15-20 alleles per locus. Therefore one must analyze more than one STR to get a more distinctive result. This is accomplished by a process called multiplexing were one must simultaneously amplify many STR loci using multiple sets of primers in a single PCR reaction. In the United States, crime labs use commercial multiplex STR kits to accomplish this process. One of these commonly used kits is the PowerPlex16 kit, manufactured by Promega, which can type all 13 of the CODIS STR loci. STR kits have been available since 1993, and multiplex STR kits were validated in 1999. CODIS which is the convicted offender database in the United States was launched by the FBI in 1998.

This figure shows chromosomal location of the 13 CODIS STR loci and Amelogenin (Amel) which is not an STR but is used to determine the sex of the suspect.

Another technique commonly used to increase the amount of DNA from a small sample is whole genome amplification (WGA), which was invented in 1992. Unlike PCR which is restricted to specific sequences flanked by primers, WGA replicates and represents the entire genomic sequence from start to finish. Although early versions of WGA were PCR based, a newer method is currently available. This method is called Multiple Displacement Amplification (MDA). The MDA technique relies on a DNA polymerase from bateriaphage ?29, which amplifies DNA isothermally at 30?C, and random hexagon primers. As ?29 polymerase replicates DNA it displaces “secondary structures” so it can produce larger fragments then PCR-based WGA.

Whole Genome Amplification. (A) Tag polymerase in PCR reaction. (B) ?29 polymerase in MDA

This means that as ?29 polymerase extends primers also it displaces DNA strands which are attached to the template strand causing a ‘hyperbranching’ mechanism, which is how the fragments are produced. The final result of MDA based WGA is a high yield of long DNA fragments which represent an entire genome.

Multiple Displacement Amplification. As ?29 polymerase replicates in the 5’ to 3’ direction it displaces the strand in front of it (as it displaced the loop in the previous figure) causing a ‘hyperbranching’ effect. This technique is referred to as the “strand displacement amplification approach” because the final amplified fragments are formed as a result of this displacement.

Since the ?29 polymerase has an error rate of 10-6, these fragments are useful for forensic evidence testing, DNA sequencing, cloning, and probe development. The MDA method can use a small sample of DNA, and can even be amplified from unpurified DNA (including cell lysates). However, the method requires non-degraded DNA, which is often found in forensic samples, to be accurately amplified. Medically the technique could facilitate genome screening for SNPs and allow one to build control samples for rare phenotypes because the amplified DNA is not statistically different from the original sample.

References

[1] M. Lynch, S. Cole, R. McNally, K. Jordan, Truth Machine: The Contentious History of DNA Fingerprinting. The University of Chicago Press, Chicago, 2008.

[2] L. Kobilinsky, T.F. Liotti, J. Oeser-Sweat, DNA: Forensic and Legal Applications, Wiley-International, Hoboken, 2005.

[3] J.T. McClintock, Forensic DNA Analysis: A Forensic Manual, CRC Press, Boca Raton, 2008.

[4] Qiagen, WGA Tutorial, http://www1.qiagen.com/Products/ByApplication/WholeGeomeAmplification/Tutorial/

[5] R.S. Lasken and M. Egholm, Whole genome amplification: abundant supplies of DNA from precious samples or clinical specimens, Trends Biotechnol. 21 (2003), 531–535.

online, Optical density, http://www.biology-online.org/dictionary/Optical_density.

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Comments (1)

Voted up. Very interesting

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