04762nam a2200181   4500001000700000100004100007101000800048200016100056210003700217215002000254300030000274330390200574606002904476606000804505606002904513700002304542701001504565390982  a20120531d2011       y0pory50      ba  aeng  aImproving the efficiency of DNA casework analysis through simple, effective, PCR-based screening methodsbRecurso eletrónicofJanice A. Nicklas, Eric Buel  a[Waterbury, VT]ced. do a.d2011  a1 CD-ROMd12 cm  aResearch report submitted [by Vermont Forensic Laboratory] to the U.S. Department of Justice - Final report of NIJ Grant # 2005-DA-BX-K003. Ficheiro de 5,77 MB em formato PDF (116 p.). Disponível também em: https://www.ncjrs.gov/pdffiles1/nij/grants/236689.pdf. Acedido a 31 de maio de 2012.  aThe forensic laboratory must continually meet the challenge of increased casework andthe criminal justice community’s demand for timely analysis of evidence. New assays that couldassess sample quality (degraded versus intact) or easily yield preliminary source information(DNA screening to differentiate victim, suspect, male, female) would allow the laboratory tofocus its energy on the most probative case samples. To achieve these goals, this grant had two major Aims: 1) development of a fast, simple profiling method for sample screening to select samples which would be most probative and 2) development of a test for sample DNA degradation state.To accomplish Aim #1, multiplex SNP assays were developed using a melting FRET technique. In this assay, two probes are present: a sensor with a perfect match to one allele (with fluorophore) but with one mismatch to the other allele and a second, anchor probe (with quencher). As PCR proceeds, fluorescence is quenched; in the melting phase, fluorescence is gained. Determination of which allele(s) is present depends on the melting temperature where the fluorescence is regained. Several assays were developed for the six color Corbett RG6000 and for other four color real-time instruments. This technique can discriminate between 95-99% of samples from different individuals. A paper describing these results is published in the Journal of Forensic Sciences. The SNP FRET assay is quite complicated in terms of number of primers and it uses biallelic SNPs which are unfamiliar to most forensic analysts. We, therefore, sought a new assay which uses multi-allelic STRs familiar to forensic analysts by attempting to adapt the melting FRET technique to STRs. While some differences were detected, the technique did not work as hoped and was abandoned. We have added an additional part to Aim #1 (AIM #1C) through a GAN: Development of a new assay for fast determination of stain donor using high resolution melting (HRM) of STRs. HRM goes beyond classical melt curve analysis by studying the melt in much finer detail using special DNA dyes such as Eva Green™. PCR products can be differentiated based on length, sequence or complementarity. In particular, it was hoped that HRM could distinguish alleles of STRs. Twenty-two forensic STRs were chosen for analysis and tested with 30-50 sample DNAs with different, known genotypes by performing real-time PCR and HRM melting in a Corbett Rotorgene RG6000. The three STRs that generated the greatest difference in melting profiles between genotypes were chosen for further study and development into an assay. The assay was then tested for reproducibility, uniformity for a known genotype, and melting profile constancy over dilution. The assay can discriminate between individuals, making it useful as a screening technique. We also performed some preliminary experiments using Plexor technology and melting of STRs which were very promising. To accomplish Aim #2, we designed a multiplex PCR with two overlapping Alu amplicons using the Plexor technology. The Plexor technology relies on the use of two alternative basepairs, isoC and isoG, which pair only to each other. The forward primer is designed with a 5’ isoC with attached fluorophore and during amplification, an isoG with attached quencher is incorporated and fluorescence is quenched due to proximity of the fluorophore and quencher. The decrease in fluorescence is monitored during PCR.Amplification of the longer Alu amplicon is only possible in non-degraded or slightly degradedDNA whereas the smaller amplicon will be amplifiable even in highly degraded DNA. Theratios of the concentrations of the two products give a quantitative measure of degradation state.The concentration of DNA measured by the long product can be used to determine input DNAfor STR analysis and the ratio predicts the amount of ski-slope observed.  aANÁLISE DE VESTÍGIOS  aADN  aPRESERVAÇÃO DA PROVA  aNICKLASbJanice A.  aBUELbEric