Quantification of the total amount of human DNA isolated from a forensic evidence item is crucial for DNA normalization prior to short tandem repeat (STR) DNA analysis and a federal quality assurance standard requirement. Previous commercial quantification methods determine the total human DNA and total human male DNA concentrations, but provide limited information about the condition of the DNA sample. The PowerQuant(®) System includes targets for quantification of total human and total human male DNA as well as targets for evaluating whether the human DNA is degraded and/or PCR inhibitors are present in the sample. A developmental validation of the PowerQuant(®) System was completed, following SWGDAM Validation Guidelines, to evaluate the assay's specificity, sensitivity, precision and accuracy, as well as the ability to detect degraded DNA or PCR inhibitors. In addition to the total human DNA and total human male DNA concentrations in a sample, data from the degradation target and internal PCR control (IPC) provide a forensic DNA analyst meaningful information about the quality of the isolated human DNA and the presence of PCR inhibitors in the sample that can be used to determine the most effective workflow and assist downstream interpretation.
ftsQ is an essential cell division gene in Escherichia coli. The ftsQ gene has been sequenced, and a presumptive open reading frame has been identified; however, no protein product has been observed (A.C. Robinson, D.J. Kenan, G.F. Hatfull, N.F. Sullivan, R. Spiegelberg, and W.D. Donachie, J. Bacteriol. 160:546-555, 1984, and Q.M. Yi, S. Rockenbach, J.E. Ward, Jr., and J. Lutkenhaus, J. Mol. Biol. 184:399-412, 1985). The ftsQ gene was isolated on a 970-base-pair EcoRI-PvuII fragment of the E. coli chromosome and used to construct a trp-lac (Ptac) transcriptional fusion in plasmid pKK223-3. The fused construct (pDSC78) complemented an ftsQ1(Ts) mutant strain in trans, restoring growth at 42 degrees C on low-salt medium. An ftsQ1(Ts) mutant strain transformed with pDSC78 appeared normal upon microscopic examination, with no indication of filamentation. The ftsQ gene product was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and two-dimensional gel electrophoresis of radiolabeled, isopropyl-beta-D-thiogalactopyranoside-induced maxicell and normal cell extracts. As predicted from the nucleotide sequence, the 970-base-pair EcoRI-PvuII fragment encoded a polypeptide of approximately 31,400 daltons. Analysis of the data obtained from pulse-chase experiments in maxicells and normal cells suggests that the FtsQ protein is stable. Most of the radiolabeled FtsQ protein from maxicells was found in the inner membrane. On the basis of available information, the prior inability to detect FtsQ can be attributed to low levels of transcription or translation rather than to proteolysis.
The PowerPlex® ESI 16 Fast, ESI 17 Fast, ESX 16 Fast, and ESX 17 Fast Systems represent faster cycling versions (50 min or less) of the PowerPlex® ESI and ESX Systems released by Promega in 2009 to accommodate the ENFSI and EDNAP groups’ call for new STR multiplexes for Europe. In addition to amplification of purified DNA samples, these new faster cycling systems allow for direct amplification from single-source blood and buccal samples deposited on FTA® and nonFTA paper as well as from SwabSolution™ extracts of buccal swabs without the need for purification and quantitation. There are no changes to the autosomal primer pair sequences in the PowerPlex® ESI Fast and ESX Fast Systems compared to the original multiplexes, and full concordance at all autosomal loci and amelogenin was observed with data generated previously with the original PowerPlex® ESI and ESX Systems. This paper describes the developmental validation study performed on these new fast systems following guidelines issued by the Scientific Working Group on DNA Analysis Methods (SWGDAM) and those of the DNA Advisory Board (DAB). Validation data demonstrate that these systems are sensitive for detecting low levels of DNA while also being capable of generating robust profiles from the high amount of input DNA present in direct-amplification samples. These systems are also tolerant to both high concentrations of PCR inhibitors as well as to slight variations in the final concentration of master mix and primer pair present in the amplification reaction that might be encountered due to pipetting error. The results of this validation study demonstrate that these systems may be used on multiple thermal cyclers and capillary electrophoresis platforms.
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