Faculty of Health Sciences & Medicine

Future Research Projects

Improved Methods for Analysis of Low Amounts of Male DNA in Forensic Samples

Supervisor

Prof Angela Van Daal
Professor of Forensic Sciences

BACKGROUND

Recent technological advances in the area of DNA profiling offer the opportunity to improve the number of samples that can be successfully analyzed. STR DNA profiling has been a standard procedure in forensic laboratories for many years now. Mitochondrial DNA analysis and Y STR typing have increased the power of DNA analysis for the forensic scientist. Methodological advances have extended this even further. Whole genome amplification (WGA) methods have aided in the development of low copy number (LCN) analysis of samples in the clinical research arena, although the impact of WGA in forensics has, up until now, been minor.  The sample detection limits of some forensic samples has recently extended with the release of the REPLI-g® Mitochondrial DNA kit. This kit uses a multiple displacement WGA method to enrich for human mitochondrial DNA with minimal contamination from nuclear DNA in a simple one-tube procedure. The same principle used in the development of this kit can be applied to the enrichment of Y chromosomal DNA, and is the subject of this proposal.

Male DNA-containing samples collected from sexual assault or homicide victims can contain very low levels of male DNA amongst a large number of female epithelial cells. This often results in failure to obtain an autosomal STR typing from the male DNA donor.  Y STR analysis has been used to overcome this problem. However there are still many instances where this approach does not work.

AIMS OF THE PROJECT

The aim of this project is to develop a method for the enrichment of Y chromosome sequences, which can then be used for Y-STR and Y-SNP analysis. This proposal aims to overcome the problems associated with low levels of male DNA in a background of female DNA by the selective amplification of the Y chromosomal genomic DNA prior to Y STR or Y SNP analysis along with incorporation of mechanisms to repair any DNA degradation in the sample. Recently technologies for the amplification of damaged total genomic DNA in a two step process: a processing reaction preparing damaged DNA for whole genome amplification and an amplification reaction has been established. This technology will be combined with the technologies for selective amplification of Y chromosomal genomic DNA.  An added advantage of this approach is that, in the case of semen containing samples, it removes the need for differential DNA extraction protocols, thereby minimizing the potential loss of sperm DNA to the epithelial cell fraction.

METHODS

The multiple displacement amplification (MDA) method of whole genome amplification is able to amplify DNA samples by up to 100,000-fold and generates microgram quantities of DNA from picogram amounts of starting template. While the use of random hexamers normally makes this a non-specific reaction, the MDA reaction can be targeted to amplify specific sequences as illustrated by the Qiagen REPLI-g® Mitochondrial DNA kit which specifically amplifies the human mitochondrial genome. The specificity of amplification using the REPLI-g® Mitochondrial DNA kit is a result of mitochondrial DNA-specific primer design especially adapted to isothermal reactions and buffer optimization. Total human cellular DNA usually contains ~ 0.1% mitochondrial DNA. After differential amplification the mitochondrial DNA fraction is enriched greater than 1x 106 fold.   We intend to apply these same principles to the development of a Y chromosome specific DNA amplification protocol.