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.
Chen
Xiao-he
XH
Southampton General Hospital, UK.
O'Dell
Sandra D
SD
Day
Ian N M
IN
D041322:Chromosomes, Human, Y; D015331:Cohort Studies; D004252:DNA Mutational Analysis / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D005260:Female; D005819:Genetic Markers; D005820:Genetic Testing / Q000379:methods*; D006801:Humans; D008297:Male; D018598:Minisatellite Repeats / Q000235:genetics*; D011110:Polymorphism, Genetic
D005819:Genetic Markers