Journal ArticleDOI
Purification of forensic specimens for the polymerase chain reaction (PCR) analysis
Atsushi Akane,Hiroshi Shiono,Kazuo Matsubara,Hiroaki Nakamura,Masanori Hasegawa,Masato Kagawa +5 more
TLDR
Purification methods of deoxyribonucleic acid (DNA) from degraded and contaminated forensic samples were investigated for polymerase chain reaction (PCR) analysis, and the amelogenin gene for sex determination could be amplified by dual PCR technique.Abstract:
Purification methods of deoxyribonucleic acid (DNA) from degraded and contaminated forensic samples were investigated for polymerase chain reaction (PCR) analysis. DNA extracted from putrefied tissue or bloodstains sometimes contained the copurified contaminant, that was identified as the porphyrin compound (hematin). When contaminated but less degraded DNA was analyzed by PCR, it was necessary to eliminate the impurity by anion exchange column chromatography or chelating resin preparation, and ultrafiltration using Centricon microconcentrators. When highly degraded DNA was analyzed, trace amounts of high molecular weight DNA was recovered by electroelution method, and then further purified by both column chromatography and ultrafiltration. From thus purified samples, the amelogenin gene for sex determination could be amplified by dual PCR technique.read more
Citations
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Journal ArticleDOI
Relief of amplification inhibition in PCR with bovine serum albumin or T4 gene 32 protein
TL;DR: Use of BSA and gp32 together offered no more relief of inhibition than either alone at its optimal level, and neither protein had any noticeable effect on amplification in the absence of inhibitors.
Journal ArticleDOI
Identification of the heme compound copurified with deoxyribonucleic acid (DNA) from bloodstains, a major inhibitor of polymerase chain reaction (PCR) amplification.
TL;DR: The heme compound found in deoxyribonucleic acid (DNA) extracted from bloodstains, which is regarded as a major inhibitor of polymerase chain reaction (PCR), was characterized in comparison with alkaline and acid hematin, histidine and ammonia hemochromogens, and globin and serum albumin hemochROMogens digested by proteinase K.
Journal ArticleDOI
Design and evaluation of Bacteroides DNA probes for the specific detection of human fecal pollution
TL;DR: Because Bacteroides spp.
Journal ArticleDOI
Development of an Alu-based, real-time PCR method for quantitation of human DNA in forensic samples.
Janice A. Nicklas,Eric Buel +1 more
TL;DR: This manuscript describes and validates a variation of this Alu-based assay using real-time PCR and SYBR Green I for quantitation that has a dynamic range of 16 ng to 1 pg, is sensitive, specific, fast, quantitative, and uses only 2 microL of sample.
Journal ArticleDOI
A quantitative PCR assay for the assessment of DNA degradation in forensic samples
TL;DR: A multiplex quantitative PCR assay has been designed to amplify target sequences of different length, which allows for the assessment of DNA degradation in samples of forensic interest, and this estimate, along with the internal control for PCR inhibition, provides a valuable tool for post-extraction sample assessment.
References
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Randall Keichi Saiki,David H. Gelfand,Susanne Stoffel,Stephen J. Scharf,Russell Higuchi,Glenn Thomas Horn,Kary B. Mullis,Henry A. Erlich +7 more
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Chelex 100 as a Medium for Simple Extraction of DNA for PCR-Based Typing from Forensic Material
Walsh Ps,Metzger Da,Higuchi R +2 more
TL;DR: The extraction of DNA from semen and very small bloodstains using Chelex 100 is as efficient or more efficient than using proteinase K and phenol-chloroform extraction and DNA extracted from bloodstain seems less prone to contain PCR inhibitors when prepared by this method.
Journal ArticleDOI
Avoiding false positives with PCR
S Kwok,Russell Higuchi +1 more
TL;DR: The exquisite sensitivity of the polymerase chain reaction means DNA contamination can ruin an entire experiment and adherence to a strict set of protocols can avoid disaster.
Journal ArticleDOI
A general method of in vitro preparation and specific mutagenesis of DNA fragments: study of protein and DNA interactions
TL;DR: These procedures, which can circumvent the need for large-scale phage or plasmid growths, preparative gel-electrophoresis and the screening of molecular clones, can facilitate the rapid study of sequence-specific interactions of proteins and DNA.
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