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Microplate array diagonal gel electrophoresis for cohort studies of microsatellite loci

Microplate array diagonal gel electrophoresis for cohort studies of microsatellite loci
Microplate array diagonal gel electrophoresis for cohort studies of microsatellite loci
After PCR amplification, we have achieved precise sizing of trinucleotide and tetranucleotide microsatellite alleles on 96-well open-faced polyacrylamide microplate array diagonal gel electrophoresis (MADGE) gels: two tetranucleotide repeats, HUMTHOI (five alleles 248-263 bp) and DYS390 (eight alleles 200-228 bp), and DYS392, a trinucleotide repeat (eight alleles 210-231 bp). A gel matrix of Duracryl, a high mechanical strength polyacrylamide derivative, and appropriate ionic conditions provide the 1.3%-1.5% band resolution required. No end-labeling of primers is needed, as the sensitive Vistra Green intercalating dye is used for the visualization of bands. Co-run markers bracketing the PCR fragments ensure accurate sizing without inter-lane variability. Electrophoresis of multiple gels in a thermostatically controlled tank allows up to 1000 samples to be run in 90 min. Gel images were analyzed using a Fluorlmager 595 fluorescent scanning system, and alleles were identified using Phoretix software for band migration measurement and Microsoft Excel to compute fragment sizes. Estimated sizes were interpolated precisely to achieve accurate binning. Microsatellite-MADGE represents a utilitarian methodfor high-throughput genotyping in cohort studies, using standard laboratory equipment.
human, non-us governmentt, minisatellite repeats, humans, methods, electrophoresis, male, laboratories, research support, dna mutational analysis, genetic screening, cohort, software, genetics, female, chromosomes, polymorphism, polyacrylamide gel, genetic, cohort studies, alleles, genetic markers, y
0736-6205
1080-1089
Chen, Xiao-He
fd93b896-d8cc-4b0e-a811-c0e6a8ff8308
O'Dell, Sandra D.
36100c24-d942-4d7d-99c0-a387a4f63644
Day, Ian N.M.
b749b30a-1f4c-40eb-af0e-a50427388b39
Chen, Xiao-He
fd93b896-d8cc-4b0e-a811-c0e6a8ff8308
O'Dell, Sandra D.
36100c24-d942-4d7d-99c0-a387a4f63644
Day, Ian N.M.
b749b30a-1f4c-40eb-af0e-a50427388b39

Chen, Xiao-He, O'Dell, Sandra D. and Day, Ian N.M. (2002) Microplate array diagonal gel electrophoresis for cohort studies of microsatellite loci. Biotechniques, 32 (5), 1080-1089.

Record type: Article

Abstract

After PCR amplification, we have achieved precise sizing of trinucleotide and tetranucleotide microsatellite alleles on 96-well open-faced polyacrylamide microplate array diagonal gel electrophoresis (MADGE) gels: two tetranucleotide repeats, HUMTHOI (five alleles 248-263 bp) and DYS390 (eight alleles 200-228 bp), and DYS392, a trinucleotide repeat (eight alleles 210-231 bp). A gel matrix of Duracryl, a high mechanical strength polyacrylamide derivative, and appropriate ionic conditions provide the 1.3%-1.5% band resolution required. No end-labeling of primers is needed, as the sensitive Vistra Green intercalating dye is used for the visualization of bands. Co-run markers bracketing the PCR fragments ensure accurate sizing without inter-lane variability. Electrophoresis of multiple gels in a thermostatically controlled tank allows up to 1000 samples to be run in 90 min. Gel images were analyzed using a Fluorlmager 595 fluorescent scanning system, and alleles were identified using Phoretix software for band migration measurement and Microsoft Excel to compute fragment sizes. Estimated sizes were interpolated precisely to achieve accurate binning. Microsatellite-MADGE represents a utilitarian methodfor high-throughput genotyping in cohort studies, using standard laboratory equipment.

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More information

Published date: May 2002
Keywords: human, non-us governmentt, minisatellite repeats, humans, methods, electrophoresis, male, laboratories, research support, dna mutational analysis, genetic screening, cohort, software, genetics, female, chromosomes, polymorphism, polyacrylamide gel, genetic, cohort studies, alleles, genetic markers, y

Identifiers

Local EPrints ID: 59570
URI: http://eprints.soton.ac.uk/id/eprint/59570
ISSN: 0736-6205
PURE UUID: 2531ed86-b54f-4cb6-9cca-08c631d63eed

Catalogue record

Date deposited: 03 Sep 2008
Last modified: 13 Mar 2019 20:30

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