Development and validation of a human DNA quantification and sex determination approach based on real time PCR followed by high resolution melting analysis

GINART SANTIAGO; ALECHINE E; CAPUTO M; CORACH D; SALA A

Kracovia

Congreso; 26th ISFG Congress International Society for Forensic Genetics; 2015

International Society for Forensic Genetics

DNA quantitation is one ofthe most crucial factors affecting the success and quality of DNA typing byPCR. The aim of this work was to develop a DNA quantification assay to be usedin routine forensic casework. It should be able to discriminate,simultaneously, the presence of male and female DNA by means of multiplex realtime PCR, followed by high resolution melting analysis (HRM),including a fluorescent intercalating dye Syto 9 due to its high sensitivity.The approach consists in theco-amplification of gene fragments common to both genders, Amelogenin ?Amel.The 6 bases pairs (bp) differencepresent in the sequences located at X chromosome and the Y counterpartgenerates a two peak pattern differing in 0.2ºC. Aiming to boost thediscrimination efficiency a human specific Y chromosome sequence (HSYCS) was also included in the reaction cocktail.The melting temperatures of this amplicon differ from Amel in 5.3-5.5ºC. Hence,it allows discriminating two peaks after HRM analysis if only male DNA ispresent in the sample or a single peak if only female template is present. Theshort length of both amplicons, 106/112 bp for Amel and 84 bp for HSYCS,facilitates quantitation and gender detection in samples potentially degradedthat characterize evidentiary material. We achieved the quantification of male,female and mixture samples with very low DNA quantities (20 pg/ul) We propose an alternativeapproach to commercial DNA quantitation kits. Our development showed to befast, highly sensitive, laborsaving and cost-effective.