Direct comparison of post-28-cycle PCR purification and modified capillary electrophoresis methods with the 34-cycle "low copy number" (LCN) method for analysis of trace forensic DNA samples.
The investigation of samples with low amounts of template DNA remains at the forefront of forensic DNA research and technology as it becomes increasingly important to gain DNA profile information from exceedingly trace levels of DNA. Previous studies have demonstrated that it is possible to obtain short tandem repeat (STR) profiles from <100pg of template DNA by increasing the number of amplification cycles from 28 to 34, a modification often referred to as "low copy number" or LCN analysis. In this study, we have optimised post-PCR purification techniques applied after only 28 cycles of PCR, as well as using modified capillary electrophoresis injection conditions and have investigated the progressive application of these enhanced approaches. This paper reviews the characteristics of the profiles obtained by these methods compared with those obtained on the same samples after 34-cycle PCR. We observed comparable sensitivity to 34-cycle PCR in terms of the number of profiles with evidence of DNA and the number of allelic peaks per profile and we noted improved peak height and area magnitude with some sample types. Certain parameters reported to be adversely affected in 34-cycle LCN investigations, such as non-donor allele peaks and increased stutter peak ratio, were reduced by this approach. There are a number of advantages for trace samples in progressing from the standard 28-cycle process to the post-PCR processing method as compared to 34-cycle PCR method, including reduced sample consumption, reduced number of PCR amplifications required, and a staged approach to sample processing and profile interpretation.
Forster
Luke
L
LGC Forensics, Middlesex, UK.
Thomson
Jim
J
Kutranov
Stefan
S
D004247:DNA / Q000032:analysis / Q000235:genetics*; D019075:Electrophoresis, Capillary / Q000379:methods; D004817:Epidermis / Q000737:chemistry; D053803:Forensic Genetics / Q000379:methods*; D006801:Humans; D008297:Male; D018895:Microsatellite Repeats / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012661:Semen / Q000737:chemistry; D012680:Sensitivity and Specificity; D013698:Templates, Genetic; D014110:Touch
D004247:DNA