This project succeeded in optimizing protocols for pre-amplification of human DNA by using whole-genome amplification (WGA). WGA is an improved isothermal whole-genome DNA amplification method that has been previously demonstrated to enable the highly sensitive amplification of bacterial DNA. It was adapted for the pre-amplification of trace human DNA to enable the improved recovery and quality of STR profiles. Reaction methods were optimized for four types of samples: 1) quantitated dilutions of de-identified intact donor genomic DNA; 2) limiting dilutions of intentionally UVC damaged genomic DNA; 3) limiting dilutions of intentionally fragmented genomic DNA ; and 4) captured intact cells from an established cell line. Project results are being transitioned for consideration to the Human Identification group at GE Healthcare. Although there are patents and patent applications for methods discussed in this report, a new patent application is being prepared on the use of Ping Pong sequence-specific amplification technologies for targeted pre-amplification of samples. Ping Pong was used to target the pre-amplification of desired STR loci. Although more work is required by qualified forensic laboratories in validating whole genome amplification from trace samples, project researchers conclude that their work has demonstrated the proposed method of sample pre-amplification could be used to increase robustness and peak heights obtained from trace or low copy number samples. In addition, if three or more intact single cells are present, complete STR profiles can be obtained by using this protocol with the AT GenomiPhi method. 12 figures and 2 tables
Molecular Tools for Low-Template DNA Analysis
NCJ Number
251935
Date Published
March 2016
Length
17 pages
Annotation
Findings and methodology are presented for a research project whose goal was to adapt two separate, highly sensitive methods for the treatment of low-template samples that would pre-amplify DNA from a few copies to an amount sufficient for quantification and robust profiling, in addition to archiving.
Abstract