A simple sequencing protocol for genotyping the <i>HLA-C</i>locus by the Sanger method

Abstract

The HLA-C gene, which belongs to the HLA superfamily (Human Leukocyte Antigen), codes for the Major Histocompatibility Complex (MHC), which plays crucial roles in the human immune system. This study aimed to develop a simple sequencing protocol by the Sanger method using fewer primers and reactions for genotyping the HLA-C gene than published protocols. The simple protocol with three primers includes one PCR reaction and two sequencing reactions. The primer set comprising SEQ ID1 and SEQ ID2 was used for the PCR reaction to specifically amplify the exon 2 – exon 3 region of the HLA-C locus, which contains the typical SNPs of each HLA-C allele. The PCR product was purified and used as a template for the sequencing reactions. Two forward primers, SEQ ID1 and SEQ ID3 were used for sequencing, in which, the SEQ ID1 forward primer is located in the intron 1 region and the SEQ ID3 forward primer is located in the intron 2 region of the HLA-C gene. Testing the simple sequencing protocol on four samples of known HLA-C genotypes showed 100% accurate results. The established Sanger sequencing protocol is simple to implement, and reduces cost and time. Thus, this protocol can be used for HLA-C sequencing for pharmacogenetic studies and applications.