Fundamentals of Forensic DNA Typing , Fundamentals of Forensic DNA Typing , کتابخانه دیجیتال جندی شاپور اهواز

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Fundamentals of Forensic DNA Typing

PCR-based DNA typing of biological evidence is now widely used in forensic analyses due to the obvious advantages of enhanced sensitivity, the ability to distinguish discrete alleles and efficacy with degraded samples. A multiplex short tandem repeat (STR) system has been previously developed which successfully co-amplifies six STR loci HUMTH01, D21S11, D18S51, D8S1179, HUMVWF31/A and HUMFIBRA (FGA) in conjunction with the X-Y homologous gene Amelogenin. This is known as the second generation multiplex system (SGM). Detection of the PCR products is undertaken on ABD 373A or 377 automated sequencers using denaturing polyacrylamide gels coupled with fluorescent-based technology. We have evaluated this system for routine forensic use and demonstrated that the technique is robust and reproducible under conditions consistent with those encountered in a forensic environment. A total of 132 stains from simulated and actual casework were analysed, together with relevant control areas and reference samples. The success rate was high with 76% of stains giving full profiles; we were also able to successfully detect and interpret mixtures. No mistyping was observed. A detailed examination of each of these profiles has assisted in the development of guidelines for casework interpretation. Although artefacts, stutter peaks and undenatured DNA were occasionally observed, these did not interfere with the accuracy of interpretation. In addition 38 samples, previously examined using the quadruplex system, were analysed with the SGM to enable a direct comparison to be made between the systems. The performance of the system with poor quality samples demonstrated its use as a rapid and powerful technique for individual identification.

The validation of a 7-locus multiplex STR test for use in forensic casework. (I). Mixtures, ageing, degradation and species studies.

Genotyping, as applied to linkage mapping, human identification, or mapping of genetic traits, mandates electrophoretic separation systems that enable a user to identify alleles with high precision to obtain a correct genotype. For 2-bp microsatellites or short tandem repeats (STRs), standard deviations of ±0.3 nucleotide are required to ensure with 99.7% probability the identity or dissimilarity of tested alleles. A complete system, consisting of commercially available laser-induced fluorescence capillary electrophoresis (ABI PRISM 310) and performance optimized polymer 4 (POP-4), was evaluated for microsatellite separations. POP-4 is a low viscosity polymer for use in uncoated fused microbore silica capillaries. It separates DNA fragments that differ in size by 1 nucleotide up to 250 nucleotides and that differ in size by 2 nucleotides for fragments up to at least 350 nucleotides in length in about 30 min. The presence of denaturants and, more importantly, operation at 60°C was mandatory for high-precision and high-resolution sizing operation. Reproducible separation performance was achieved in excess of 100 injections per capillary with resulting standard deviations in the range of 0.04 to 0.17 nucleotide. Comparative sizing of known CEPH (Centre d’Etudes du Polymorphisme Humaine) samples performed at 22 independent test sites showed the usefulness of the system for genotyping with standard deviations of 0.24 nucleotide, or better.

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High-Precision Genotyping by Denaturing Capillary Electrophoresis

Short tandem repeat (STR) loci are routinely employed for individual identification. WE have examined the performance and reproducibility of a highly informative co-amplification system containing the tetranucleotide STR loci: HUMVWFA31/A, HUMTH01, D20S85, D8S1179, HUMFIBRA, D21S11, and D18S51, in conjunction with the amelogenin sex test, in addition to a modified system omitting the locus D20S85. Polymerase chain reaction (PCR) products were fluorescently detected on an automated sequencer and automatically sized against an internal size standard by Genescan software. Both systems were routinely able to type 500 pg of undegraded DNA. At DNA concentrations between 50-500 pg, partial profiles were produced, but no allelic drop-out was observed. Balanced amplification of all loci occurred over a wide range of DNA concentrations from 50 pg to 10 ng. Alteration of reagent concentrations and cycling parameters from optimal resulted in variation in the efficiency of individual locus amplification relative to the other loci within the system. This was also observed at high ionic strength or extreme pH. However, at all reagent concentrations and conditions, allelic drop-out was not observed. These multiplex systems have potential in both routine forensic and intelligence database applications.

Validation of highly discriminating multiplex short tandem repeat amplification systems for individual identification

Validation and casework application of a Y chromosome specific STR multiplex.

A system of four short tandem repeat loci (HUMVWF31A, HUMTH01, HUMF13A1, and HUMFES/FPS) has been tested in co-amplification with forensic (post-mortem and post-coital) DNA samples. Semiautomated DNA typing was employed to analyze polymerase chain reaction (PCR) products formed by extension of primers labeled with a fluorescent dye at the 5'-terminus. Most DNA extracts could be typed, although a few required the addition of bovine serum albumin or a pretreatment by ultrafiltration in order to obtain sufficient signal for typing. Balanced signals for the alleles were obtained frequently across the loci, although preferential amplifications of HUMTH01 was observed often with the forensic samples. Band splitting due to nontemplate nucleotide addition to the blunt ends of the amplimers was frequently detected for the DNA extracted from the forensic samples. A data-base was constructed for the African-American population and compared with a Caucasian database. Few differences were observed across the two populations, except at the locus HUMTH01. The fluorescence-based system facilitates large-scale databasing, because the PCR products run off the gel, allowing more than one set of samples to be analyzed per run. Polyacrylamide gel reuse did not diminish genotyping accuracy.

Forensic applications of a rapid, sensitive, and precise multiplex analysis of the four short tandem repeat loci HUMVWF31/A, HUMTH01, HUMF13A1, and HUMFES/FPS

Charles A. Badger

Papers

Forensic applications of a rapid, sensitive, and precise multiplex analysis of the four short tandem repeat loci HUMVWF31/A, HUMTH01, HUMF13A1, and HUMFES/FPS