limited quantities of DNA

A common challenge with forensic casework is the recovery of limited quantities of DNA from evidentiary samples. Within the past few years a new DNA enrichment technology has been developed known as whole genome amplification (WGA). WGA involves a different DNA polymerase than the TaqGold enzyme commonly used in forensic DNA analysis. WGA amplifies the entire genome using random hexamers as priming points. The WGA enzymes work by multiple displacement amplification (MDA), which is sometimes referred to as rolling circle amplification. MDA is isothermal with an incubation temperature of 30 ° C and requires no heating and cooling like PCR. Qian(Valencia, CA) and Sigma-Aldrich (St. Louis, MO) both offer phi29 DNA polymerase cocktails for performing WGA. The kit sold by Qian is called REPLI-g while Sigma Aldrich’s kit is GenomePlex. Yields of 4 – 7 μ g of amplified genomic DNA are possible from as little as 1 ng of starting material. The phi29 enzyme has a high processivity and can amplify fragments of up to 100 kb because it displaces downstream product strands, enabling multiple concurrent and overlapping rounds of amplification. In addition, phi29 has a higher replication fidelity compared to Taq polymerase due to 3 – 5 proofreading activity. While all of these characteristics make WGA seem like a possible solution to the forensic problem of limited DNA starting material, studies have found that stochastic effects at low levels of DNA template prevent WGA from working reliably ( Schneider et al., 2004 ). Allele dropouts from STR loci were observed at 50- and 5-pg levels of starting material ( Schneider et al. , 2004 ) just as are seen with current low copy number DNA testing (see Chapter 14). Work with ‘ molecular crowding ’materials such as polyethylene glycol, where the amount of DNA is enriched in localized areas of a sample, has shown improved success with STR typing from low amounts of DNA ( Ballantyne et al. , 2006 ). While it is possible that WGA may play a limited role in enriching samples for archiving purposes that are in the low nomogram range, it will probably not be the end-all solution to low copy number DNA samples.
Sources: Ballantyne , K. N. , et al. ( 2006 ) . Molecular crowding increases the amplification success of multiple displacement amplification and short tandem repeat genotyping . Analytical Biochemistry, 355 , 298 – 303 . http://www.qiagen.com/Products/wholeGenomeAmplifi cation/ http://www.sigmaaldrich.com/life-science/molecular-biology/ automation/whole-genomeamplifi cation.html Schneider , P. M. , et al. ( 2004 ) . Whole genome amplifi cation — The solution for a common problem in forensic casework? Progress in Forensic Genetics 10 , ICS 1261 , 24 – 26 .