Multiplex amplification and typing procedure for the loci D1S80 and amelogenin

Forensic Science Research Unit, FBI Laboratory, FBI Academy, Quantico, Virginia 22135, USA.
Journal of Forensic Sciences (Impact Factor: 1.16). 08/1996; 41(4):660-3.
Source: PubMed


A method has been developed that enables multiplex amplification and simultaneous typing of the loci D1S80 and amelogenin using discontinuous polyacrylamide gel electrophoresis and silver staining. The protocol is sensitive, simple, rapid, and relatively inexpensive. The results of the multiplex analysis of the D1S80 and amelogenin loci were comparable to those obtained when each locus was analyzed individually. A small validation study was undertaken to evaluate the forensic applicability of this multiplex system. The data demonstrate that DNA exposed to a variety of environmental insults yields reliable multiplex typing results.

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    ABSTRACT: In this study, a technique was developed to separate by capillary electrophoresis (CE) the widely varying DNA fragment sizes produced by a multiplex polymerase chain reaction (PCR) amplification of the loci D1S80 and amelogenin. Experiments were performed to analyze different buffer systems and obtain optimal resolution for the separation. A matrix composed of two different molecular weights of the same polymer was constructed to separate the DNA fragments with baseline resolution, and a cubic spline fit was used to estimate the size of DNA fragments over 350 base pairs. Over 100 samples were examined to demonstrate the rapid, robust and precise characteristics of this CE system. An average relative standard deviation of 0.3% was obtained for the sizing of the D1S80 alleles in these samples. DNA from mixed body fluid samples, samples subjected to environmental insult, and D1S80 sequence variants were also typed successfully. These results demonstrate that CE is a viable method for analysis of D1S80 and amelogenin forensic DNA samples.
    No preview · Article · Sep 1996 · Electrophoresis
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    ABSTRACT: When aligning the human amelogenin X (AmelX)-gene with the human amelogenin Y (AmelY)-gene, 19 regions of absolute homology ranging in size from 22 to 80 bp, 5 deletions located on AmelX from 1 to 6 bp and 5 deletions 1-183 bp long located on AmelY can be observed. The regions of absolute homology are used to design primer sets which span deletions of the X- and/or Y-chromosome. The PCR products generated on the X- and Y-homologues differ in size. New regions of deletions and newly designed primers are described to facilitate the integration of the amelogenin sex test into multiplex PCR reactions.
    No preview · Article · Feb 1997 · Deutsche Zeitschrift für die Gesamte Gerichtliche Medizin
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    ABSTRACT: A genetic locus D1S80 containing a variable number of tandem repeats (VNTR) has been used extensively in forensic analysis and paternity testing. In the current research, the D1S80 locus was amplified using polymerase chain reaction (PCR) technology and the alleles detected using a high sensitivity infrared (IR) fluorescence automated DNA sequencer. IR-labeled amplification products were generated using oligonucleotide primers which were covalently linked to an infrared fluorescent dye (IRD41) at the 5'-end. Human genomic DNA (1.0 ng or less) isolated from blood and various simulated forensic samples was successfully amplified using this technology. Allelic bands were detected by incorporation of the IR fluorescent dye into PCR products. Both Long Ranger and polyacrylamide denaturing gels permitted clear resolution of individual alleles that differ by only one repeat unit. In the smaller gels a separation distance of only 15 cm allowed separation of the alleles in less than 2 h from sample loading to visualization. This system combines IR fluorescence chemistry and laser technology thus eliminating the need for post-electrophoretic gel handling for the detection of D1S80 alleles. Real-time detection is valuable for immediate visualization of the data and the alleles are displayed as familiar autoradiogram-like images which can also be analyzed by computer. By loading a 64-lane gel twice it is possible to type at least 120 samples in 1 day using a single gel.
    No preview · Article · Jun 1997 · Forensic Science International
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