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

Abstract

Corresponding author: Junin 956 Piso 7 (1113, Buenos Aires Argentina). Tel./fax: 00541149648281/8282. DNA quantitation is one of the most crucial factors affecting the success and quality of DNA typing by PCR. The aim of this work was to develop a DNA quantification assay to be used in routine forensic casework. It should be able to discriminate, simultaneously, the presence of male and female DNA by means of multiplex real time PCR, followed by high resolution melting analysis (HRM), including a fluorescent intercalating dye Syto 9. The approach is co-amplified fragments of a gene common to both genders, Amelogenin-Amel and a specific sequence of the human Y chromosome (HSYCS) whose melting temperature differs from Amel in 5.3-5.5ºC. Hence, it allows discriminating two peaks after HRM analysis if only male DNA is present in the sample or a single peak if only female template is present. The short length of both amplicons, 106/112 bp for Amel and 84 bp for HSYCS, facilitates quantitation and gender detection in degraded samples that characterize evidentiary material. We achieved the quantification of male, female and experimentally mixed samples with very low DNA quantities (20 pg/ul). We propose an alternative approach to commercial DNA quantitation kits. Our development showed to be fast, highly sensitive, laborsaving and cost-effective. IntroductionHuman DNA quantification in forensic samples is one of the most important factors underlying the efficiency and success of PCR-based genotyping. DNA input between 0,5 and 2,0 ng may result, in case no inhibitors are present in the sample, in complete profile with the available commercial kits. Since men commit most of the violent crimes, male DNA detection in the evidence is fundamental for determining the best strategy for the investigation [1]. Currently, there are many commercial kits for the quantification of DNA and detection of male DNA simultaneously, shown to be highly sensitive and robust, but their use greatly increases the cost of analysis in forensic casework. Alternative systems to commercial kits have been previously reported [2,3] although the detection systems employed (such as TaqMan probes) exceeds the complexity of intercalating dyes selected for our development.Our goal was to develop a human DNA quantization method able to discriminate the presence of male and female human DNA by means of multiplex real time PCR, followed by high resolution melting analysis (HRM), including a fluorescent intercalating dye Syto 9, due to its DNA double strand saturation ability, resulting in higher sensitivity. Based on previous investigations in our lab, we selected one Amelogenin gene fragment as candidate to quantify human DNA. Presence of male DNA amplifies a 112 bp fragment and female DNA amplifies a 106 bp amplicon. After HRM we observed two melting peaks respectively that differ in 0,2°C. While this difference in melting behavior allow discriminating the sex identity from DNA, is not optimal to ensure correct resolution of forensic mixed samples. Aiming to...

Development of a nested real time PCR/high resolution melting assay for human T-cell lymphotropic viruses types 1 and 2 (HTLV-1 and 2) identification

“…DNA-free samples and blanks were also included as controls.The first amplification round was performed using primers POS and POA (Table 1). PCR reaction mixture was: 5 ng total genomic DNA (quantified as described by Ginart et al 2015), 20pM of each primer, 0Á16 mM dNTPs, 25 mM syto9, 1 U of GoTaqâ polymerase (Promega, Madison, WI) and 5X Go Taq reaction buffer in a total volume of 25 ll. The cycling conditions were: 2 min denaturation at 95°C; 30 cycles consisting of 30 s at 94°C, 40 s at 50°C and 20 s at 72°C; followed by 2 min extension at 72°C in a real-time PCR equipment RotorGene 6000 (Corbet Inc., Sidney, Australia).…”

“…DNA was extracted from 500 ll of cadaveric blood by CTAB method (Corach et al 1995). Total genomic DNA quantification was performed as described by Ginart et al (2015). The presence of the HTLV-1 or HTLV-2 genome was determined in all samples by the real-time PCR protocol followed by high resolution melting described above.…”

The Usefulness of qPCR Data for Sample Pre-Assessment and Interpretation of Genetic Typing Results

“…For its sensitivity, specificity, accuracy, precision, and multiplexing ability, quantitative real-time PCR (qPCR) is the gold standard for human DNA quantification in current forensic practices [ 7 , 8 , 9 , 10 , 11 , 12 ]. qPCR is a PCR-based technique in which the presence of fluorescent reporter molecules allows for the real-time detection of PCR amplicons.…”