Kidd (SLC14A1 rs1058396) Genotyping Performed by PCR-Restriction Fragment Length Polymorphism Using a General-Purpose Agarose Gel and Lithium Borate Buffer

Abstract

Background: Kidd (SLC14A1 rs1058396) genotyping is an important test to prevent delayed hemolytic transfusion reactions and hemolytic disease of the fetus or newborn. The PCR-restriction fragment length polymorphism (RFLP) technique using the MnlI restriction enzyme is not used widely because of the need for a polyacrylamide gel. PCR-RFLP was performed by electrophoresis with general-purpose agarose gel, and lithium borate buffer (LBB) was developed as a replacement for polyacrylamide gel. Methods: Seventy-two venous blood samples were collected randomly and used in this study. A 3% agarose gel containing 1 µg/mL of ethidium bromide and 1 mM LBB, and a high-voltage (300 V) were used to separate the short-length restriction fragments (72 bp, 51 bp, 36 bp, and 21 bp). The PCR-RFLP results were confirmed by PCR-direct sequencing. Results: Target restriction fragments could be easily discriminated. The results obtained with the PCR-RFLP were completely in agreement with the results of PCR-direct sequencing. Conclusion:The PCR-RFLP using a general-purpose agarose gel and LBB is an accurate and reliable assay for Kidd genotyping.