Pressure-based alkaline lysis with immunocapture, a method for enhanced recovery in differential extraction.

A pressure-based protocol for differential extraction has been optimized to provide a rapid and selective alternative to conventional differential extraction techniques with the advantage of an increased recovery of genomic DNA. The protocol involves treating cotton swabs containing mixtures of sperm and vaginal epithelial cells with two sets of pressure treatment using a Barocycler® NEP 2320 in alkaline conditions. This first step quantitatively and selectively removes female epithelial cells. The cotton swab is removed and further treated with alkali at 95°C for the removal of sperm cell DNA. The resultant solution provides a clean male profile at a 20:1 cell ratio. Furthermore, the inclusion of a pretreatment step involving immunomagnetic cell capture of the female cells, permits nearly complete isolation of male sperm cells at cell ratios of up to 200:1 female to male cells.

Genotyping horse epithelial cells from fecal matter by isolation of polymerase chain reaction products.

AIM: To show that application of the polymerase chain reaction (PCR) method modified for amplification of a low-copy number DNA samples, ie, the isolation of PCR products (IPCRp), would represent improvement in obtaining genotypes from a fecal DNA compared with previously used genotyping methods. METHODS: The DNA from the horse fecal matter was extracted by modified Qiagen DNA Stool Mini Kit protocol. Following the extraction, the DNA genotypes from fecal samples were obtained by the most powerful PCR amplification method, the IPCRp. The IPCRp-based multiplex kit amplified biotin-labeled strands were captured on streptavidin-coated plates, where everything but the dye-labeled target sequence was washed, eliminating all the background noise, released, and run on a genotyping instrument in a single-strand configuration. RESULTS: The IPCRp-based multiplex kit (6 loci) revealed equine DNA full genotype profiles, ie, appearance of all six loci, when sampled from fresh feces in 87% of the samples and partial genotype profile (appearance of one to five loci) in 13% of the samples, for a total of 100% genotyping success rate. CONCLUSION: These results indicate that the IPCRp amplification method, coupled with the Qiagen DNA Stool Mini Kit extraction can maximize the likelihood of obtaining horse DNA genotypes from fecal samples.

Increasing detection of polymerase chain reaction (PCR) by isolation of PCR products (IPCRp).

AIM: To develop a method for enhanced polymerase chain reaction (PCR) product detection. METHODS: During the PCR, the double-stranded product is generated with fluorescent dye on one strand, and biotin on the other strand. The product is captured on the streptavidin-coated plates with high efficiency (IPCRp). Washing of the all unamplified compounds, including dye-labeled unincorporated primers, follows the PCR. The targeted dye-labeled PCR product is released by denaturation and loaded on the detection platform. RESULTS: After the application of the IPCRp, the resulting product is highly concentrated targeted dye-labeled single-stranded DNA, free of the unincorporated primers and other PCR artifacts. The strength of the signal of the IPCRp product on detection platform is two- to five-fold higher than the strength of the signal of the conventional PCR product. CONCLUSION: The IPCRp procedure can be accomplished in less than 20 minutes. Efficient isolation of the PCR products has two steps, washing and denaturation. It can increase the yield of targeted PCR product and increase the sensitivity of the detection platform.

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.