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Title: A LOW-COST HIGH THROUGHPUT POLYACRYLAMIDE GEL ELECTROPHORESIS SYSTEM FOR GENOTYPING WITH MICROSATELLITE DNA MARKERS

Author
item Cregan, Perry
item WANG, D - MICHIGAN STATE UNIVERSITY
item CARLSON, S - MICHIGAN STATE UNIVERSITY
item WARD, R - MICHIGAN STATE UNIVERSITY
item DIERS, B - UNIVERSITY OF ILLINOIS
item SHI, J - CHINA

Submitted to: Crop Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/10/2003
Publication Date: N/A
Citation: N/A

Interpretive Summary: Microsatellite or simple sequence repeat (SSR) DNA markers can be used for a variety of purposes in plant genetic and plant improvement research including the identification of breeding lines with specific disease resistance or quality traits. However, using conventional methods to assay SSR markers is expensive and the throughput is relatively limited. The objective of this paper was to report the development of a new low-cost, high-throughput system for the detection SSR DNA markers using polyacrylamide gel electrophoresis. This electrophoresis system permits the detection of two sets of SSR markers from 100 plant samples at one time in less than two hours. With five of the gel systems, a skilled person can simultaneously process 10 gels and obtain at least 1000 data points in five hours. The cost per gel is estimated at about $2.60, so the cost per data point is less than $0.03. This low cost and high throughput system should allow the large scale application of SSR marker technology in plant breeding programs thereby allowing the rapid incorporation of new genes for diseased resistance, product quality, etc. into new breeding lines. This information should be useful to academic scientists as well as plant breeders in the commercial sector.

Technical Abstract: Microsatellite DNA markers are widely used in genetic research but the genotyping cost with this marker system is high and the throughput is limited with conventional methods. The objective of this paper is to introduce a low-cost, high-throughput system developed in our laboratories for the detection of amplification products from microsatellite markers by non-denaturing polyacrylamide gel electrophoresis. This system is capable of separating DNA fragments that differ by as little as two base pairs. The electrophoresis unit holds two 100-sample gels vertically and has a rotating base for easy gel access during loading with a multi-channel pipette. This allows standards and samples from a 96-well plate to be analyzed on a single gel. DNA samples are stained during electrophoresis by ethidium bromide in the running buffer. In addition, one of the gel plates is made of UV-transparent glass so gels can be photographed immediately after electrophoresis without disassembling the gel-plate sandwich. Electrophoresis runs are generally less than two hours. With five of the gel systems, a skilled person can simultaneously process 10 gels and obtain at least 1000 data points in five hours. Sample throughput, using this system, can be further increased by multiplexing 2 or more samples per well. The cost per gel, excluding PCR cost, is currently estimated at about $2.60, so the cost per data point is less than $0.03. This system has been used successfully with soybean [Glycine max (L.) Merr.] and wheat (Triticum aestivum L.) and could be a valuable tool for other researchers employing microsatellite markers.