Differentiating ABO Genotypes by Using qPCR Genotyping Methods

Presenter Information

Kristirose Renwick, Minnesota State University, Mankato

Location

CSU 253/4/5

Start Date

4-4-2011 11:00 AM

End Date

4-4-2011 12:30 PM

Student's Major

Chemistry and Geology

Student's College

Science, Engineering and Technology

Mentor's Name

Theresa Salerno

Mentor's Department

Chemistry and Geology

Mentor's College

Science, Engineering and Technology

Description

The objective of this research was to develop a qPCR (quantitative polymerase chain reaction) genotyping assay to identify the common ABO genotypes. The ABO blood system has A, B, and O phenotypes but genotypes include the common variant alleles of A101, A201, B101, O101, O201 and O303. Previously, a qPCR genotyping method was developed that involved the single nucleotide polymorphisms (SNPs) at positions 261 and 297. At nucleotide 261 types A and B have a guanine while O101 and O201 have the guanine deleted from the sequence. At the 297 SNP, type A and O101 have an adenine while type B and O201 have a guanine. Ambiguity was found in identifying some heterozygote genotypes making it necessary to use another SNP to distinguish between A/O201 and B/O101 genotypes and between O303 and B alleles. The SNP 930 was chosen because it allows the identification of the B101 allele; the B allele has unique nucleotide adenine at this position. The DNA samples were obtained using a DNA purification kit, and their concentrations were estimated using a spectrophotometer at a wavelength of 260. The qPCR primers and probes were designed using software from Applied Biosystems. The method was validated with samples of known genotypes. It was tested with samples previously showing ambiguities. By combining all qPCR genotyping methods for the three SNPs most tested variants were distinguished, and the ambiguities were relieved.