Purification of Forensic Specimens for the Polymerase Chain Reaction (PCR) Analysis

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.

Protocol for extraction of genomic DNA from swine solid tissues

“…Higher concentrations were obtained from the liver, kidney and brain tissues, similar to those obtained by Atmadja et al (1995) for human tissues. The OD260/OD280 ratio values satisfied those suggested by Sambrook et al (1989), ≥ 1.8, which were higher than those obtained by Akane et al (1993) and Atmadja et al (1995).…”

A quantitative PCR assay for the assessment of DNA degradation in forensic samples

“…To assess the sensitivity of the IPC to inhibition, a series of artificially inhibited DNA extracts were examined using the triplex qPCR assay. Twenty identical tubes of non-degraded DNA were treated with different amounts of hematin, a known PCR inhibitor [24], such that the final concentrations of hematin in the standard 20 mL qPCR assays ranged from 0 to 160 mM. Each sample was assayed in duplicate with the nuTH01-nuCSF-IPC triplex qPCR assay, and the results are summarized in Table 4.…”

“…Features making qPCR particularly attractive for forensic applications are: (i) qPCR assays can be designed to quantify specific genomes of interest; (ii) the assays can be sensitive enough to detect only a few copies or even a single copy of target DNA; (iii) qPCR dynamic detection ranges readily span the roughly three orders of magnitude (e.g., 30 pg to 30 ng of nuclear DNA) needed for most forensic applications; and (iv) the experimental protocols for real-time qPCR quantitations are straightforward, labor-saving, and amenable to automation. Moreover, the use of target-specific detection www.elsevier.com/locate/forsciint Forensic Science International 158 (2006) [14][15][16][17][18][19][20][21][22][23][24][25][26] chemistries (e.g., TaqMan 1 [3] or Molecular Beacon [4] probes) makes it possible to design multiplex, real-time qPCR assays that can simultaneously quantify more than one target in a sample, offering the possibility for saving time, labor, and extracted DNA.…”

Synergy between DNA polymerases increases polymerase chain reaction inhibitor tolerance in forensic DNA analysis

“…However, the success rate of diagnostic PCR analysis is lowered by the presence of PCRinhibitory substances [2], a problem especially prominent in forensic DNA analysis due to the often complex sampling environment at crime scenes [3][4][5]. Recently, we reported that 12% of volume crime saliva stains with adequate DNA amounts analyzed at our laboratory still produced negative DNA profiles due to the presence of inhibitors [6].…”