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United States Department of Agriculture

Agricultural Research Service

Title: A Denaturing Polyacrylamide Gel System for High Throughput Analysis of Simple Sequence Repeat (Ssr) DNA Markers

Authors
item Cregan, Perry
item Quigley, Charles

Submitted to: Plant Genome Conference Proceedings
Publication Type: Abstract Only
Publication Acceptance Date: March 18, 1997
Publication Date: N/A

Technical Abstract: The analysis of SSR markers requires the ability to distinguish small differences in the length of DNA fragments. This requirement often necessitates the use of DNA sequencing gels with 32P labeling to obtain sufficient resolution. In addition, double stranded fragments amplified from certain SSR alleles tend to associate and form higher molecular weight products when separation is performed under non-denaturing conditions. In these instances a clear determination of allele size and/or genetic status (homo- vs. heterozygote) is difficult. Furthermore, in the context of a plant breeding program one is faced with the necessity of high sample throughput. To meet these requirements and constraints, a denaturing polyacrylamide gel electrophoresis system was designed that can readily distinguish SSR alleles that vary by as little as 4 basepairs, does not require the use of radio labeled compounds, and which is run on a microtiter plate format with a multi-channel pipette used for both set-up of polymerase chain reactions (PCR) as well as gel loading. Using this system a total of 192 individual samples can be genotyped on one gel. A 32 tooth comb or a 64 tooth comb is used. After a pre-running, four sets of 32 or 64 samples are loaded every 40 to 60 min followed by electrophoresis at 60 watts constant power. Gels are stained using the single strand DNA-specific stain SYBR Green I and photographed with on a standard UV transilluminator.

Last Modified: 7/22/2014
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