Microplate Array Diagonal Gel Electrophoresis for Cohort Studies of Microsatellite Loci. (May 2002)

• Record Type: Journal Article
• Authors: Chen, Xiao-he; O'Dell, Sandra D.; Day, Ian N.M.
• Is Part Of: Biotechniques. Volume 32:Number 5(2002)
• Page Start: 1080
• Page End: 1089
• Publication Date: 2002-05
• Subjects: Biology, Experimental -- Periodicals; Molecular biology -- Periodicals; Medical technology -- Periodicals; Biology, Experimental; Medical technology; Molecular biology; Clinical Laboratory Techniques -- Periodicals; Research -- Periodicals; Medical Laboratory Science -- Periodicals; Periodicals; Electronic journals; 570
• DOI: 10.2144/02325rr03
• Languages: English
• ISSNs: 0736-6205

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, HUMTH01 (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 TM, 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 TM 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 FluorImager ® 595 fluorescent scanning system, and alleles were identified using Phoretix TM 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 method for high-throughput genotyping in cohort studies, using standard laboratory equipment.