Use of coagulation factor XIII (F13) gene as an internal control for normalization of genomic DNA’s for HLA typing

Abstract

Genomic DNA (gDNA) obtained from whole blood samples is a critical element for genomic research and clinical diagnosis. PCR efficiencies of the targeted genes like HLA-A, -B, -C, DPB1 and DRB1 using such isolated gDNAs were variable in spite of having similar amounts of gDNA taken for PCR. We addressed such PCR variabilities by normalizing the gDNA’s using an internal control of human coagulation factor XIII that was found to be variable with all samples and did not correlate with the observed A260 nm readings. The PCR and Q-PCR methodologies for the human coagulation factor XIII have been optimized, and the advantages of normalizing gDNA preparations based on F13 copy numbers have been discussed. This method will serve as a suitable choice to be used in laboratories and research centres, particularly when dealing with a large number of samples for the next-generation sequencing purposes, and in forensic labs with limited sample availability. Method name: Use of coagulation factor XIII (F13) gene as an internal control for normalization of genomic DNA’s for HLA typing, Keywords: DNA, Electrophoresis, Extraction, Factor XIII, Human blood, PCR, Spectrophotometry, Spin-column