NCJ Number
219246
Journal
Journal of Forensic Sciences Volume: 52 Issue: 4 Dated: July 2007 Pages: 820-829
Date Published
July 2007
Length
10 pages
Annotation
This study evaluated different post-PCR (polymerase chain reaction) purification methods in an attempt to improve the analytical sensitivity of standard STR (short tandem repeat) DNA typing.
Abstract
Using the Qiagen MinElute silica column, the purified PCR product produced a fourfold increase in fluorescent signal intensity over the unpurified product. Further, by adding the entire concentrated purified PCR product, a 19-fold increase in signal intensity was achieved. Post-PCR purification with the MinElute column can greatly enhance the sensitivity of the PCR process, obtaining full profiles down to 20 pg input template DNA and generating significant data down to 5 pg without increasing amplification cycles. This purification method is simple, inexpensive, and can be accomplished in approximately 15 minutes. By adjusting the volume of eluate and the amount of purified product injected, the sensitivity of this technique can be controlled. The study used a 28-cycle PCR to evaluate various methods of post-PCR purification for their effects on the sensitivity of fluorophore-based allelic detection subsequent to capillary electrophoretic separation. Initially, four post-PCR purification methods were assessed by a comparison of profile integrity and relative fluorescent signal intensity. These included two filtration methods (using the Millipore Corporation Microncon-50 and Montage PCR filters), binding to a silica gel membrane (using the Qiagen MinElute PCR Purification Kit), and enzymatic hydrolysis of primers and nucleotides (using ExoSAP-IT from USB). The amplified product was purified by using filtration, silica gel membrane, and enzyme mediated hydrolysis purification techniques. They were evaluated for their effect on fluorescent allelic signal intensity. 7 tables, 6 figures, and 22 references