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

AIM: To describe the development and performance of the new horse genotyping kit.

METHODS: Highly discriminatory 17-Plex horse genotyping kit was designed by adding the fifth dye to the StockMarks kit for genotyping horses and taking advantage of the new instrument platforms. This was accomplished by using a new set of five fluorescent dyes developed by Applied Biosystems (DS-31), with four of the dyes used to label the forward amplification primers (6-FAM, VIC, NED, and PET) in each primer set.

RESULTS: The new equine kit contained five extra loci (ASB17, LEX3, HMS1, CA425, and ASB23) in addition to the 12 original loci (VHL20, HTG4, AHT4, HMS7, HTG6, HMS6, HTG7, HMS3, AHT5, ASB2, HTG10, and HMS2) recommended by the International Society for Animal Genetics. The kit performed well on different instrument platforms (ABI PRISM 377, 310, and 3100 instruments) and across wide ranges of DNA template concentrations (1-10 ng). These 17 loci were combined and amplified in a single polymerase chain reaction (PCR) cycle, which dramatically improved the power of statistical tests for pedigree analysis while reducing the time and work required to perform such tests. An in-lane size standard labeled with the fifth dye (LIZ) provided accurate size determination for genotyping.

CONCLUSION: The new 17-Plex horse kit, designed to improve the laboratory efficiency by genotyping more markers in a shorter time, has 17 primer sets labeled with new fluorescent dyes, which can be amplified in one PCR cycle and genotyped in one run on a high-throughput instruments.