DNA profiling of sperm cells by using micromanipulation and whole genome amplification

Abstract

The differential extraction method is based on separate lysis of vaginal cells and spermatozoa and was designed to prevent mixed DNA profiles from intimate swabs. However, DNA from the victim can still be present in the sperm fraction, and the suspect’s DNA cannot be identified when only minute amounts of spermatozoa are present. Moreover, differential extraction is not effective when swabs contain sperms from more than one individual. Mixed profiles could ideally be overcome by analysing single spermatozoa. However, current multiplex STR kits are not yet sensitive enough to generate DNA profiles from single cells.The aim of this study was to develop a method that enables DNA profiling of up to a single sperm cell. Spermatozoa were isolated through micromanipulation. Spermatozoa were lysed and their DNA was pre-amplified by whole genome amplification (WGA) to generate sufficient template for PCR. To these ends, several WGA methods were first tested on different amounts of genomic DNA (gDNA) and assessed for allele recovery, allele drop-out (ADO) and allele drop-in (ADI). The best WGA method was selected for use on cell material.The REPLI-g method turned out as the only protocol increasing the sensitivity of DNA profiling. Results of WGA performance on gDNA as well as multiple and single cells will be presented.