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E A Benzinger

Publications and source records attributed to E A Benzinger.

8 recordsLinked to original sources
PubMed1999

Long PCR for VNTR analysis.

The Polymerase Chain Reaction (PCR) has revolutionized the analysis of DNA from a variety of sources. With its sensitivity and ability to amplify degraded DNAs and small quantities of samples, coupled with fast turn-around-time, PCR is often the analytical method of choice for DNA profiling in forensic laboratories. RFLP methods, while requiring larger amounts of high molecular weight DNA and needing approximately 6-8 weeks of analytical time, still provide a higher power of discrimination per locus than that achieved using the loci currently available for PCR. The combination of both RFLP and PCR would be advantageous for some applications. A new technique, Long PCR, allows for the effective amplification of long DNA targets from approximately 0.5 kb to > 20 kb of genomic DNA. Currently, several Long PCR systems are commercially available. Using a Taq/Pyrococcus DNA polymerase enzyme system and DNA isolated from bloodstains, we have successfully amplified 1-20 ng of Chelex-extracted DNA, an amount commonly used in Amp-FLP technology. The robustness of Long PCR in comparison to RFLP was also examined through the use of partially degraded blood samples. Long PCR was then used to amplify both D2S44 and D5S110 RFLP loci. Although all D2 and D5 alleles were detected, the larger alleles were amplified at significantly lower levels than the smaller alleles.

Amino Acid Sequence↗
PubMed1998

An illustrated guide to RFLP troubleshooting.

A large number of reagents and steps are required for restriction fragment length polymorphism (RFLP) analysis, which at times make determining the cause of any observed anomaly difficult. Troubleshooting problems in RFLP analysis is difficult and often the exact cause of a problem cannot be determined. In this paper a collection of controlled experiments detail the consequences of a number of human or materials problems. Although the focus is on forensic applications, this troubleshooting guide will be helpful to anyone employing Southern analysis.

Artifacts↗
PubMed1997

Products of partial digestion with Hae III. Part 1. Characterization, casework experience and confirmation of the theory of three-, four- and five-banded RFLP pattern origins using partial digestion.

The sizes of Hae III partial digestion products at D1S7, D2S44, D4S139, D5S110, D10S28, and D17S26 were evaluated in experimentally generated partial digestions of liquid blood DNA. The partial digestion products were highly predictable, suggesting a very high level of sequence conservation in regions flanking variable number tandem repeat (VNTR) blocks. Partial digestion bands associated with three-or-more-banded patterns were also characterized. Partial digestion of three-banded patterns can be used to determine whether the extra bands arise due to internal Hae III sites in the VNTR block and to identify hidden three-banded patterns. Partial digestion products from forensic casework also conformed to size expectations. Presumed partial digestion bands from 27 forensic samples were compared to the experimentally generated data. The causes of partial digestion are examined and recommendations for interpreting forensic DNA evidence exhibiting partial digestion products are given.

Blood Stains↗
PubMed1997

Products of partial digestion with Hae III. Part 2. Quantification.

The base pair size of the excess DNA in the smallest three partial digestion bands for the variable number of tandem repeat loci D1S7, D2S44, D4S139, D5S110, D10S28, and D17S26 has been quantitatively evaluated using data obtained from intentional partial digestion of liquid blood DNA. Restriction fragment length polymorphism (RFLP) measurement characteristics specific to the performing laboratory were evaluated from that laboratory's historical K562 cell line control data. The expected size of the excess DNA is estimated as the weighted mean of the differences between the measured size of the partially digested bands and the fully digested band, with the weights predicted using knowledge of RFLP measurement characteristics. Confidence limits are developed for evaluating whether the size differences among a set of RFLP band multiplets observed in pristine samples are consistent with those expected from partial digestion. The base pair size of excess DNA for partials observed in evidentiary samples appears to be somewhat less than that from pristine samples.

Blood Stains↗
PubMed1996

Validation of probe EFD52 (D17S26) for forensic DNA analysis.

Validation studies that meet TWGDAM (The Working Group on DNA Analysis Methods) and CAC (California Association of Criminalists) guidelines for RFLP (restriction fragment length polymorphism) analysis were performed with the DNA probe EFD52 (D17S26). These studies demonstrate that the probe EFD52 is suitable for forensic casework. No unexpected DNA banding patterns were obtained from controlled studies examining various tissues, sample consistency over many gels, mixtures of body fluids, various substrates, various contaminants and non-human DNA sources. Of all the animal DNAs tested, only one higher primate yielded a single band to EFD52 hybridization. The sensitivity of EFD52 was shown to be comparable to that of other forensic probes. Population frequency distribution tables were prepared from over 4000 alleles and two-locus studies were conducted on nine forensically useful probes. Black, White, Hispanic and Lumbee Indian populations were found to be in Hardy-Weinberg and linkage equilibrium. Comparisons between victim blood standards and epithelial fractions of mixed strains from sexual assault cases were used to demonstrate the robustness of the EFD52 probe in forensic casework.

Asian People↗
PubMed1995

Evaluation of the specificity of alkaline phosphatase-conjugated oligonucleotide probes for forensic DNA analysis.

Current methods of forensic DNA profiling by restriction fragment length polymorphism analysis rely on radioactive detection of DNA. The use of radioactive isotopes is complicated, expensive and requires elaborate safety precautions. Recently, non-radioactive detection methods involving the use of alkaline phosphatase-conjugated oligonucleotide probes have become available. These probes differ from most 32P-labeled probes in that they are synthetic oligonucleotides, whereas the 32P-labeled probes are purified plasmid inserts from cloned VNTR regions. Because of this difference, it is possible that the specificity of the non-isotopic probe will differ from that of the 32P-labeled probe. This study compares the specificity of the two types of probes by parallel analysis of a set of DNA samples, including a subset of relatively small alleles, at the loci D1S7, D2S44, D4S139, D10S28, and D17S26. We found that although the alkaline phosphatase detection method is slightly less sensitive than 32P detection, the AP-conjugated oligonucleotide probes tested have specificity comparable to, and are appropriate and suitable substitutes for, 32P-labeled plasmid inserts.

Alkaline Phosphatase↗
PubMed1995

Time course and inhibitors of Hae III digestion in the forensic laboratory.

Forensic restriction fragment length polymorphism (RFLP) profiling has become standardized in the United States and Canada to the extent that all federal, state and local forensic laboratories employ the restriction enzyme Hae III. Forensic DNA samples are sometimes resistant to digestion with Hae III. This problem is typically ascribed to sample characteristics and is dealt with through prolonged incubation with Hae III, repeated incubations and/or re-extraction. We determined and extensively tested the maximum useful duration of Hae III incubation for several types of samples. We also examined the effect of common DNA extraction reagents and laboratory procedures on subsequent restrictability of DNA. No benefit whatsoever was found to continuing digestion reactions past 1 hour. Certain laboratory reagents were found to inhibit Hae III activity at extremely low concentrations whereas others were found to have little effect on the success of the restriction digestion. Significantly, no contaminating reagent or other laboratory procedure was found to cause loss of Hae III specificity, whether enzyme activity was compromised or not. The results of this study allow the streamlining of the RFLP procedure and provide a basis for understanding how laboratory procedures may adversely affect Hae III activity and DNA digestion.

Bacteriophage phi X 174↗