Development of a 17-plex microsatellite polymerase chain reaction kit for genotyping horses.

This research covers the development of a more efficient method for genotyping horses using a 17-plex microsatellite polymerase chain reaction (PCR) kit. This new kit improves lab procedures by allowing all 17 loci to be genotyped in just one run, significantly reducing both time and manual labor.

Methodology and Development of the Kit

• The researchers enhanced the currently existing StockMarks kit for genotyping horses by adding the fifth dye to it. This led to the formation of their advanced 17-plex horse genotyping kit.
• This enhancement took advantage of the latest instrument platforms and a newly designed set of five fluorescent dyes by Applied Biosystems (DS-31). Out of these, four – 6-FAM, VIC, NED, and PET – were used to label the forward amplification primers in each primer set.
• The developed kit included five new loci (ASB17, LEX3, HMS1, CA425, and ASB23) on top of the 12 original loci (VHL20, HTG4, AHT4, HMS7, HTG6, HMS6, HTG7, HMS3, AHT5, ASB2, HTG10, and HMS2), officially recommended by the International Society for Animal Genetics.
• It’s noteworthy that all these 17 loci could be combined and amplified in a single PCR cycle, which led to increased efficacy of statistical tests for pedigree analysis, as well as substantial reduction in time and effort.

Testing and Results

• The team conducted tests which showed good performance of the new kit across several instrument models (ABI PRISM 377, 310, and 3100) and a wide range of DNA template concentrations (1-10 ng).
• A precise size determination for genotyping was facilitated by an in-lane size standard labeled with the fifth dye – LIZ.

Conclusion

• The developed 17-plex horse kit has valuable implications for genetic research in horses. By genotyping more markers in a shorter time frame, it significantly enhances laboratory efficiency.
• The main highlight of the kit is the 17 primer sets labeled with the new fluorescent dyes. These enable a single high-throughput PCR cycle for amplifying the markers and genotyping them in a single run.