Abstract...
DNA profiles can be obtained from fabrics where a person has made direct contact with clothing. A standard approach is to cut out a section of the fabric and then use a commercially available method to extract and isolate the DNA. Alternative methods to isolate DNA include the use of adhesive tape to remove traces of cellular material from the fabric prior to extraction. We report on a process to obtain full DNA profiles using direct amplification from a range of fabrics. The absence of an extraction step both reduces the opportunity for contamination and reduces the loss of DNA during the extraction process, increasing the sensitivity of the process of generating a DNA profile. The process does not require the use of commercially available extraction kits thus reducing the cost of generating a DNA profile from trace amounts of starting material. The results are in part dependent upon the nature of the fabric used to which the DNA has been transferred.
Origine:fsigenetics.com
Visualizzazione post con etichetta DNA Forensic. Mostra tutti i post
Visualizzazione post con etichetta DNA Forensic. Mostra tutti i post
martedì 10 novembre 2009
Developmental validation of the PowerPlex® 16 HS System: An improved 16-locus fluorescent STR multiplex
Abstract....
STR multiplexes remain the cornerstone of genotyping forensic samples. The PowerPlex® 16 HS System contains the core CODIS loci: D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D21S11, CSF1PO, FGA, TH01, TPOX, and vWA. Additional loci amplified in the multiplex reaction are the sex-determinant locus, amelogenin, and two pentanucleotide STR loci, Penta D and Penta E. The PowerPlex® 16 HS System is an updated version of the PowerPlex 16® System; while the primers and dyes remain unchanged, it introduces an enhanced buffer system that includes hot-start Taq DNA polymerase and ensures robust performance. Due to the modification of the reaction mix, a multi-laboratory developmental validation study was completed to document performance capabilities and limitations for the revised assay. Data within this validation was generated by eight laboratories and served as the basis for the following conclusions: genotyping of single-source samples was consistent across a large range of template DNA concentrations with most laboratories obtaining complete profiles at 62.5pg. Mixture analyses showed that over 90% of minor alleles were detected at 1:9 ratios. Optimum amplification cycle number was ultimately dependent on the sensitivity of the detection instrument and could be adjusted to accommodate a range of DNA template concentrations. Reaction conditions including volume and annealing temperature as well as the concentrations of primers, Taq DNA polymerase, and magnesium were shown to be optimal and able to withstand moderate variations without affecting multiplexed STR amplification. Finally, data from non-probative samples and concordance studies showed consistent results when comparing the PowerPlex® 16 HS System with the PowerPlex® 16 System as well as other commercially available systems.
Origine:fsigenetics.com
STR multiplexes remain the cornerstone of genotyping forensic samples. The PowerPlex® 16 HS System contains the core CODIS loci: D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D21S11, CSF1PO, FGA, TH01, TPOX, and vWA. Additional loci amplified in the multiplex reaction are the sex-determinant locus, amelogenin, and two pentanucleotide STR loci, Penta D and Penta E. The PowerPlex® 16 HS System is an updated version of the PowerPlex 16® System; while the primers and dyes remain unchanged, it introduces an enhanced buffer system that includes hot-start Taq DNA polymerase and ensures robust performance. Due to the modification of the reaction mix, a multi-laboratory developmental validation study was completed to document performance capabilities and limitations for the revised assay. Data within this validation was generated by eight laboratories and served as the basis for the following conclusions: genotyping of single-source samples was consistent across a large range of template DNA concentrations with most laboratories obtaining complete profiles at 62.5pg. Mixture analyses showed that over 90% of minor alleles were detected at 1:9 ratios. Optimum amplification cycle number was ultimately dependent on the sensitivity of the detection instrument and could be adjusted to accommodate a range of DNA template concentrations. Reaction conditions including volume and annealing temperature as well as the concentrations of primers, Taq DNA polymerase, and magnesium were shown to be optimal and able to withstand moderate variations without affecting multiplexed STR amplification. Finally, data from non-probative samples and concordance studies showed consistent results when comparing the PowerPlex® 16 HS System with the PowerPlex® 16 System as well as other commercially available systems.
Origine:fsigenetics.com
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