High-throughput real-time PCR-based genotyping without DNA purification
1 Department of Molecular Genetics, Microbiology, and Immunology, UMDNJ-Robert Wood Johnson Medical School, Piscataway, NJ, USA
2 Reproductive Medicine Associates of New Jersey, Morristown, NJ, USA
3 Bonei Olam, Center for Rare Jewish Genetic Disorders, 1755 46th St, Brooklyn, NY, 11204, USA
4 RMA of NJ ATTN, Anastasia Fedick 111 Madison Ave, Suite 100, Morristown, NJ, 07960, USA
BMC Research Notes 2012, 5:573 doi:10.1186/1756-0500-5-573Published: 19 October 2012
While improvements in genotyping technology have allowed for increased throughput and reduced time and expense, protocols remain hindered by the slow upstream steps of isolating, purifying, and normalizing DNA. Various methods exist for genotyping samples directly through blood, without having to purify the DNA first. These procedures were designed to be used on smaller throughput systems, however, and have not yet been tested for use on current high-throughput real-time (q)PCR based genotyping platforms. In this paper, a method of quantitative qPCR-based genotyping on blood without DNA purification was developed using a high-throughput qPCR platform.
The performances of either DNA purified from blood or the same blood samples without DNA purification were evaluated through qPCR-based genotyping. First, 60 different mutations prevalent in the Ashkenazi Jewish population were genotyped in 12 Ashkenazi Jewish individuals using the QuantStudio™12K Flex Real-Time PCR System. Genotyping directly from blood gave a call rate of 99.21%, and an accuracy of 100%, while the purified DNA gave a call rate of 92.49%, and an accuracy of 99.74%. Although no statistical difference was found for these parameters, an F test comparing the standard deviations of the wild type clusters for the two different methods indicated significantly less variation when genotyping directly from blood instead of after DNA purification. To further establish the ability to perform high-throughput qPCR based genotyping directly from blood, 96 individuals of Ashkenazi Jewish decent were genotyped for the same 60 mutations (5,760 genotypes in 5 hours) and resulted in a call rate of 98.38% and a diagnostic accuracy of 99.77%.
This study shows that accurate qPCR-based high-throughput genotyping can be performed without DNA purification. The direct use of blood may further expedite the entire genotyping process, reduce costs, and avoid tracking errors which can occur during sample DNA purification.