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Jon Wilson M.S.F.S. Valerie Fuller Ph.D. Gifty Benson M.S.F.S. D.D.S. Denise Juroske M.S.F.S. Eric Duvall M.S.F.S. Jun Fu Ph.D. Jane Pritchard M.T. C.L.S. Robert W. Allen Ph.D. 《Journal of forensic sciences》2010,55(4):1050-1057
Abstract: A method is described for the quantitation of total human and male DNA. Q‐TAT utilizes end‐point, multiplex polymerase chain reaction (PCR) amplification of the amelogenin and SRY loci to quantify DNA and incorporates a cloned nonhuman template to detect PCR inhibition. Standard curves of fluorescence from amelogenin or SRY amplicons were generated from amplification of known amounts of NIST traceable SRM‐female or SRM‐male DNA. Curves showed good linearity up to 500 pg of SRM‐template (R2 > 0.99) and reliably estimated total and male DNA content in casework samples. The nonhuman pRLnull template included in each PCR was a sensitive indicator of known PCR inhibitors including EDTA, hemin, blue denim dye, and humic acid. Finally, the SRY amplicon was a sensitive indicator of male DNA and, in mixtures, could reliably estimate male DNA present in an excess of female DNA. The Q‐TAT multiplex is a reliable quantitation method for forensic DNA typing. 相似文献
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Abstract: Amplification of DNA from aged or degraded skeletal remains can be a challenging task, in part due to naturally occurring inhibitors of the polymerase chain reaction. PCR inhibitors may act by inactivating a polymerase itself, or compete with or bind other reaction components, although various polymerases may be differentially susceptible to such insult. In this study, ten thermostable polymerases from six bacterial species were examined for their ability to amplify DNA in the presence of bone‐derived or individual PCR inhibitors. Two polymerases, one from Thermus aquaticus and one from Thermus thermophilus, showed lower susceptibility to inhibition from bone, while polymerases from Thermus flavus were highly susceptible. Addition of bovine serum albumin improved the activity of most of the enzymes. Taken together, the results indicate that thermostable DNA polymerases have different susceptibility to bone‐derived PCR inhibitors, and that those most often used in forensic laboratories may not be optimal when working with DNA from skeletal remains. 相似文献
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目的研究多重置换扩增(multiple displacement amplification,MDA)在含抑制物检材中的抗抑制能力,与磁珠法纯化检材相比较,证明其在法医学中的应用及意义。方法将不同浓度血红素和腐殖酸与样本DNA进行混合,分为MDA处理组、磁珠法处理组和空白对照组,PCR-STR单基因座D3S1358扩增联合聚丙烯酰胺凝胶电泳检测,并应用Amp F詛STR誖IdentifilerTMPlus试剂盒联合毛细管电泳检测。结果血红素质量浓度大于1 ng/μL或腐殖酸质量浓度大于0.1 ng/μL时,空白对照组经单基因座STR检测不能得到扩增产物;磁珠法处理组血红素质量浓度大于100 ng/μL或腐殖酸浓度大于1 ng/μL时,不能够得到扩增产物;MDA处理组各浓度抑制物均能成功扩增,完全不受抑制物影响。结论 MDA技术可消除血红素及腐殖酸的抑制作用,其抗抑制能力优于磁珠法纯化DNA,具有一定的法医学应用意义。 相似文献
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Forensic analysts routinely encounter samples containing DNA mixtures from male and female contributors. To obtain interpretable Short Tandem Repeat (STR) profiles and select the appropriate STR analysis methodology, it is desirable to determine relative quantities of male and female DNA, and detect PCR inhibitors. We describe a multiplex assay for simultaneous quantification of human and human male DNA using the ribonuclease P RNA component H1 (RPPH1) human target and the sex determining region Y (SRY) male-specific target. A synthetic oligonucleotide sequence was co-amplified as an internal PCR control. Standard curves were generated using human male genomic DNA. The SRY and RPPH1 assays demonstrated human specificity with minimal cross-reactivity to DNA from other species. Reproducible DNA concentrations were obtained within a range of 0.023-50 ng/μl. The assay was highly sensitive, detecting as little as 25 pg/μl of human male DNA in the presence of a thousand-fold excess of human female DNA. The ability of the assay to predict PCR inhibition was demonstrated by shifted IPC Ct values in the presence of increasing quantities of hematin and humic acid. We also demonstrate the correlation between the multiplex assay quantification results and the strength of STR profiles generated using the AmpF?STR®PCR Amplification kits. 相似文献
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Survival of Very High Blood Alcohol Concentration Without Consequential Damage in a Patient Without a Previous Substance Use Disorder
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Kathrin Malejko M.D. Heiko Graf M.D. Maximilian Gahr M.D. 《Journal of forensic sciences》2016,61(4):1155-1157
Intoxications with alcohol may lead to death depending on (maximum) blood alcohol concentration (BAC) and accompanying factors such as liver function, tolerance, and comedication. Death may occur due to ethanol‐induced respiratory depression and/or aspiration of gastric content (due to an impaired gag reflex); thus, securing of the airway and ventilation is occasionally necessary. A case of a 58‐year‐old female patient with depression who demonstrated a very high BAC of 8.68 gm/L (0.868%) following ingestion of large amounts of alcohol with suicidal intent is presented. Intubation and ventilation were life‐saving, and the patient did not develop any physical or consequential damage. As the patient had not regularly used alcohol or any other psychotropic agent, tolerance could be ruled out. This case emphasizes the necessity of rapid securing of the airway in patients with alcohol intoxication and respiratory depression and, furthermore, illustrates the large interindividual differences regarding ethanol susceptibility. 相似文献
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