ABO Genotyping using a Multiplex Single-base Primer Extension Reaction

Hyun, Jungwon; Chang, Ho Eun; Heo, Se Ran; Song, Sang Hoon; Park, Kyoung Un; Song, Junghan; Han, Kyou Sup

Abstract

BACKGROUND: ABO genotyping is being widely used in the case of ABO discrepancies and in forensic medicine. We have designed a method using a multiplex single-base primer extension reaction that has allowed us to detect six single nucleotide polymorphism (SNP) sites in the ABO gene and to determine ABO genotypes.
METHODS
Genomic DNA was isolated from the peripheral blood of 75 unrelated Korean subjects. Exon 6 containing nucleotides 261 and 297 and exon 7 containing nucleotides 703, 802, 803 and 1059 were amplified using two pairs of primers. Using the products as templates, a multiplex single-base primer extension reaction was performed with six typing primers of different lengths for the six SNP sites. These reactions were performed on a PTC-200 thermal cycler (MJ Research, Waltham, MA, USA) using the SNaPshot multiplex kit (Applied Biosystems, Foster City, CA, USA), and the products were analyzed using an ABI 3130xl Genetic Analyzer (Applied Biosystems).
RESULTS
The ABO genotypes determined by this method (75/75) all matched the genotypes that were determined by the use of the polymerase chain reaction using sequence-specific priming (PCR-SSP). We analyzed the peak pattern detected at each of the six SNP sites for each sample. For the smaller-sized primers, peaks were shifted to the right-side compared with the expected site and for the larger-sized primers peaks was close to the expected site. In addition, the coefficients of variation (CVs) of the smaller-sized primers were higher than the CVs of the larger-sized primers.
CONCLUSIONS
We are able to detect six SNP sites in the ABO gene and to determine ABO genotypes using a multiplex single-base primer extension reaction.