|Quigley, Charles - Chuck|
Submitted to: DNA Markers Protocols Applications and Previews
Publication Type: Book / Chapter
Publication Acceptance Date: 1/14/1997
Publication Date: N/A
Interpretive Summary: The use of DNA markers in plant improvement is often hindered by the technical complexity required for each analysis of the DNA marker. This is true in the case of simple sequence repeat (SSR) DNA markers which often require the use of a complex analysis apparatus and radioactive compounds to detect alternative marker types. The objective of this work was to define a system of SSR marker analysis that was technically less demanding than the standard system and which did not use any radioactive compounds. Such a system was defined and should be of particular use to geneticists interested in determining SSR marker size in large numbers of plants or animals. The use of this system would be most appropriate in plant and animal breeding programs.
Technical Abstract: The analysis of simple sequence repeat (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 or 33P labeling. 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 is difficult. Furthermore, in the context of a plant breeding program one is faced with the necessity of high sample throughput. Here we demonstrate a number of alternative systems to meet these analysis needs including a denaturing polyacrylamide gel electrophoresis system that was designed to readily distinguish SSR alleles that vary in length 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. As currently configured, a 35 (width) x 30 (length) cm, 1.5 mm thick gel (6% acrylamide, 5.6M urea, 30% formamide, 1X TAE), and a 32 (0.9 cm center to center) or a 64 tooth comb (0.45 cm center to center) is used. Four sets of 32 or 64 samples are loaded sequentially followed by electrophoresis. Gels are stained using the single strand DNA-specific stain SYBR-Green I and photographed with a SYBR-Green photographic filter on a standard UV transilluminator.