Full Length Research Paper

A new electrophoresis technique to separate microsatellite alleles*

Xinwang Wang^1,2, Timothy A. Rinehart³, Phillip A. Wadl¹, James M. Spiers³, Denita Hadziabdic¹, Mark T. Windham¹ and Robert N. Trigiano¹*

¹Entomology and Plant Pathology, the University of Tennessee, Knoxville, TN 37996, U.S.A.

²Texas AgriLife Research and Extension Center. Dallas, TX 75252, U.S.A.

³USDA-ARS Southern Horticultural Laboratory, 810 Highway 26 West, Poplarville, MS 39470 U.S.A.

*Corresponding author. E-mail: rtrigian@utk.edu. Tel: 1-865-974-0221. Fax: 1-865-974-4744.

Accepted 9 February, 2009

Analysis of large numbers of SSR (simple sequence repeats: microsatellites) reactions can be tedious, time-consuming and expensive. The objective of this study was to report a new electrophoresis method to analyze and visualize SSR data quickly and accurately and compare it to the ability of four other electrophoresis methods.  Individual PCR reactions consisting of DNA from several Cornus florida L. (flowering dogwood) cultivars and two SSR primer pairs were assembled for analysis using the following three methods: agarose gel, polyacrylamide gel and QIAxcel System. Two separate PCR reactions consisting of the same components plus a fluorescent-labeled primer were set up for analyses using the CEQ^TM 8000 Genetic Analysis System and ABI 3130xl DNA Sequencer. These five electrophoretic methods were assessed for advantages and disadvantages. Polyacrylamide gels had highest resolution of alleles, whereas agarose gels had the lowest. However, with both separation media, it was difficult to score the size of alleles. Capillary electrophoresis with the CEQ^TM 8000 Genetic Analysis System and ABI 3130xl DNA Sequencer easily separated products and determined allelic size, but was more expensive than electrophoresis using either agarose or polyacrlamide gels. The QIAxcel System had lower resolution than CEQ^TM 8000 Genetic Analysis System and ABI 3130xl DNA Sequencer.  However, QIAxcel System was rapid and cost effective compared to the two widely used capillary sequencers, and also provided a computer generated gel image.  For researchers in small to intermediate-sized laboratories, the QIAxcel System using a twelve channel, sieving-gel cartridge is an affordable device for SSR assays used for mapping and population diversity analysis.

Key words: SSR assay, capillary electrophoresis, polyacrylamide gel, Cornus florida.