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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...

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Short tandem repeats (STRs) are the markers of choice for purposes of human forensic identification because of their considerable degree of polymorphism. This variability may occasionally become a challenge for the analyst when a new variant invades the allele distribution range of the neighboring locus. We present here a novel variant at locus D1S1656 showing a molecular length of 211.32 bp corresponding to 21.3 repeating units. This variant is superimposed on the D12S391 locus when the Global Filer™ (Thermo Fisher Scientific Inc., USA) is used, invading the shortest allele range and being assigned as Off-ladder (OL). In contrast, typing with PowerPlex® Fusion (Promega Corp., Madison, USA) overlaps the D2S441 locus, generating an allele that is recognized as 8. In both cases the invasion was observed as a tri-allele pattern. The results were confirmed by DNA re-extraction and typing with the two independent commercial megaplexes described above. The variant was detected during analysis of a reference sample throughout a paternity exclusion case. This is the first time this variant has been described and reported to the NIST STR Base. Since D1S1656 is included in widely-used multiplex kits from several vendors -Biotype (Dresden, Germany) Qiagen (Venlo, The Netherlands), Promega (Madison, USA) and Thermo Fisher (Foster City, CA, USA) -it would then be recommendable that other forensic labs be aware of this new micro-variant in dealing with similar interpretation challenges and that the kit producers take this fact into account in designing new multiplex kits.