Thermal Cycling Parameters

A wide range of PCR cycling protocols have been used for various molecular biology applications. To serve as an example of PCR cycling conditions

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.

Molecular Staging (New Haven, CT) and Amersham Biosciences (Piscataway, NJ) both offer phi29 DNA polymerase cocktails for performing WGA. The kit by Molecular Staging is called REPLI-g™ while Amersham's kit is GenomiPhi™. Yields of 4-7 |ig 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 pg and 5 pg levels of starting material (Schneider et al. 2004) just as are seen with current low-copy number DNA testing (see Chapter 7). While it is possible that WGA may play a limited role in enriching samples for archiving purposes that are in the low ng range, it will probably not be the end-all solution to low copy number DNA samples.

Sources:

Schneider, P.M. et al. (2004) Whole genome amplification - the solution for a common problem in forensic casework? Progress in Forensic Genetics 10, Elsevier Science, ICS 1261, 24-26. Hosono, S. et al. (2003) Unbiased whole-genome amplification directly from clinical samples. Genome Research, 13, 954-964. Dean et al. (2002) Comprehensive human genome amplification using multiple displacement amplification. Proceedings of the National Academy of Sciences USA, 99, 5261-5266. Lizardi, P.M. (2000) Multiple displacement amplification, U.S. Patent 6124120.

See also http://www.rubicongenomics.com; http://www.the-scientist.com/ yr2003/aug/tools2_030825.html; http://www.the-scientist.com/yr2003/ janZtools_030113.html

D.N.A. Box 4.1 Whole genome amplification commonly used by forensic DNA laboratories, Table 4.3 contains the parameters used with the GenePrint® STR kits from Promega Corporation and the AmpF/STR® kits from Applied Biosystems. The primary reason that PCR protocols vary is that different primer sequences have different hybridization properties and thus anneal to the DNA template strands at different rates.

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