An immobilized sequence-specific oligonucleotide (SSO) probe system consisting of 16 SSO probes that detect sequence polymorphisms within five regions of the mtDNA control region was used to investigate the frequency of heteroplasmy in human mtDNA. Five regions of hypervariable region II (HVII) of the control region were studied in blood-, muscle-, heart-, and brain-tissue samples collected from 43 individuals during autopsy. An initial search for heteroplasmy was conducted by use of the SSO probe system. Samples in which multiple probe signals were detected within a region were sequenced for the HVII region, to verify the typing-strip results. The frequency of heteroplasmy was 5 of 43 individuals, or 11.6%. The frequency of heteroplasmy differed across tissue types, being higher in muscle tissue. The difference in the frequency of heteroplasmy across different age groups was statistically significant, which suggests that heteroplasmy increases with age. As a test for contamination and to confirm heteroplasmy, the samples were sequenced for the HVI region and were typed by use of a panel of five polymorphic nuclear markers. Portions of the tissues that appeared to be heteroplasmic were extracted at least one additional time; all gave identical results. The results from these tests indicate that the multiple sequences present in individual samples result from heteroplasmy and not from contamination.
Saliva and saliva-stained materials were examined as potential sources of deoxyribonucleic acid (DNA) for DNA analysis and identity testing. In this paper, the authors demonstrate that DNA was isolated and DNA banding patterns suitable for DNA typing were obtained from fresh saliva and various saliva-stained materials, such as envelopes, buccal swabs, gags, and cigarettes. Furthermore, DNA and DNA banding patterns were obtained from actual forensic evidentiary samples containing mixed saliva/semen stains. The DNA banding patterns obtained from saliva or saliva-stained material were indistinguishable from the patterns obtained from blood or hair from the same individual. Intact DNA was readily isolated and DNA banding patterns were obtained from saliva stored at −20°C and dried saliva stains stored under varying conditions. We conclude that saliva and saliva-stained material can be good sources of DNA for analysis and for DNA typing in certain forensic settings.
The QTrap liquid chromatography-tandem mass spectrometry (LC-MS-MS) by Applied Biosystems was investigated as an adjund to enzyme immunoassay (EIA) for the rapid detection of drugs in blood. Thus, a procedure used to identify drugs in whole blood by EIA was extended to LC-MS-MS analysis. A multiple reaction monitoring (MRM) database of over 100 drugs was constructed to analyze for those drugs commonly observed in postmortem toxicology cases. The QTrap method provided for a scan time of only 2.8 s to produce both an MRM and an enhanced product ion scan. Various validation and developmental steps of the method are presented, as well as a concordance study as a final means of validation. This study was conducted to compare the effectiveness of the QTrap versus conventional extraction methods and gas chromatography-MS for the identification of drugs in 95 postmortem samples. The more than 400 drug results in this study showed 100% concordance between the two techniques.
The observed interlaboratory standard deviation (SD) associated with the restriction fragment length polymorphism (RFLP) measurement of DNA fragment size is a predictable function of the observed mean band size (MBS). For DNA fragments of size 1,000 base pairs (bp) to the largest resolved component of commonly used "sizing ladder" calibration materials (about 20,000 bp), the variation in the sizing data from the Technical Working Group on DNA Analysis Methods (TWGDAM)-sponsored "Large Fragment Study" is well-described by SD = 7.5 (1 + MBS/19 500)7.1. This sizing variability arises from a 0.1-0.4% SD in the relative positions of sample and calibration bands among electrophoretic gels. Statistically significant sizing differences do exist for bands above 10,000 bp among laboratories that use different calibration materials. The Large Fragment Study was efficiently accomplished through the use of a designed set of DNA samples, requiring but one gel in each of 20 participating laboratories.
Deoxyribonucleic acid (DNA) was isolated from a number of spongy and compact human bone tissue specimens, and the yield was estimated on a “per milligram of starting tissue” basis. DNA was, in addition, isolated from a number of corresponding blood and bone tissue specimens. Spectrophotofluorometry and ethidium bromide visualization on minigels were used to estimate the quantity and degree of degradation of DNA. The DNA from several blood-bone pairs is shown to give concordant restriction fragment length polymorphism (RFLP) typing results by two different typing protocols with five different singlelocus probes. DNA from several additional blood-bone pairs is shown to give concordant results for human leucocyte antigen (HLA)-DQα phenotypes following polymerase chain reaction (PCR) amplification and hybridization to specific allele-specific oligonucleotide (ASO) probes, and for the variable numbers of tandem repeats (VNTR) length polymorphisms 3′ to the human apolipoprotein B (APOB) gene following PCR amplification with specific primers and analysis of the products by electrophoresis and ethidium bromide visualization.
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