ABO genotyping by single strand conformation polymorphism--using CE.

This study describes a novel method of ABO genotyping using amplicons generated by PCR-SSCP and separated by CE. We describe four amplicons with sizes of 112, 121, 123 and 160 bp based on SSCP analysis. These amplicons are amplified from exons 6 and 7 and include polymorphic sites of cDNA 261, 297, 467, 526, 646, 657, 681, 703, 1061 and 1096 generated from the ABO gene. The positions of the SSCP alleles, when rescaled using SSCP pattern as the mobility values based on an internal standard ranged, ranged from 146 to 210. The mobility variations of alleles with 1-4 SNPs within the same amplicons were from 1.88 to 12.01. These wide mobility differences allow for unambiguous allele determination. The use of different fluorescent dyes allows for the identification of two fragments with overlapping size ranges. The validity of the test was confirmed based on the SD of the mobility values from the eleven fragments of allelic ladder and it never exceeded 0.37. The mean values of the mobility variation between the samples and the ladders for the four amplicons ranged only from 0.05 to 0.39. This method was shown to successfully identify alleles A(1), A(1v), B, O(1), O(1v) and O(2) in tested samples. Since the largest amplicons are only 160 bp, this method is ideal for the fast screening ABO genotypes from trace, or seriously, degraded samples and may be valuable in clinical transfusion or forensic applications.