Whole genome amplification of degraded and nondegraded DNA for forensic purposes

Abstract

Degraded DNA is often analyzed in forensic genetics laboratories. Reliable analysis of degraded DNA is of great importance, since its results impact the quality and reliability of expert testimonies. Recently, a number of whole genome amplification (WGA) methods have been proposed as preamplification tools. They work on the premise of being able to generate microgram quantities of DNA from as little as the quantity of DNA from a single cell. We chose, investigated, and compared seven WGA methods to evaluate their ability to "recover" degraded and nondegraded DNA: degenerate oligonucleotide-primed PCR, primer extension preamplification PCR, GenomePlex™ WGA commercial kit (Sigma), multiple displacement amplification, GenomiPhi™ Amplification kit (Amersham Biosciences), restriction and circularization-aided rolling circle amplification, and blunt-end ligation-mediated WGA. The efficiency and reliability of those methods were analyzed and compared using SGMPlus, YFiler, mtDNA, and Y-chromosome SNP typing. The best results for nondegraded DNA were obtained with GenomiPhi and PEP methods. In the case of degraded DNA (200 bp), the best results were obtained with GenomePlex which successfully amplified also severely degraded DNA (100 bp), thus enabling correct typing of mtDNA and Y-SNP loci. WGA may be very useful in analysis of low copy number DNA or degraded DNA in forensic genetics, especially after introduction of some improvements (sample pooling and replicate DNA typing).

Fig. 1

SGMPlus profiles of triple PEP preamplifications of the same DNA extract and pooled products. PEP was run with 0.25 ng of template. Numbers indicate the percentage of successfully amplified alleles. a, b, c SGMPlus profiles of single, independent PEP reactions. d SGMPlus profile of pooled products

Fig. 2

Comparison of SGMPlus profiles of DNA obtained from male muscle tissue. a Nondegraded, native DNA; b DNA degraded to ca 200 bp; c, d, e degraded DNA preamplified by using GenomePlex amplification of 1, 10, and 100 ng, respectively. Asterisk indicates stutters (n-4 fraction)

Fig. 3

Comparison of HVII regions of nondegraded mtDNA (a) and degraded mtDNA preamplified with GenomePlex (b). The box marks the presence of the A/G heteroplasmy