Killer cell immunoglobulin-like receptors (KIR) typing by DNA sequencing

Abstract

DNA sequencing is a powerful technique for identifying allelic variation within the natural killer cell immunoglobulin-like receptor genes. Because of the relatively large size of the KIR genes, each locus is amplified in two or more overlapping segments. Sanger sequencing of each gene from a preparation containing one or two alleles yields a sequence that is used to identify the alleles by comparison with a reference database.

Figure 1

Amplification of overlapping amplicons covering the KIR2DL1 coding region sequence. KIR genes have eight to nine exons. PCR amplification primers are designed to generate two or more overlapping amplicons. The figure shows the three amplicons, A, B, and A2, that cover the coding sequence of the KIR2DL1 gene. If the sample does not contain KIR2DS1, the laboratory needs only to generate the A and B amplicons for sequencing as described in Table 1. Amplicon A will allow the sequence determination from nucleotide 11 of exon 1 through nucleotide 632 of exon 5; amplicon B will cover nucleotide 332 in exon 4 through the last nucleotide of exon 9. If the sample contains the KIR2DS1 gene, the laboratory will perform instead three amplifications generating amplicons A, B, and A2. The A2 amplicon will contain only KIR2DL1 and will provide the sequence covering nucleotide 1 in exon 1 through nucleotide 330 of exon 4. The A amplicon which contains DNA from both KIR2DL1 and KIR2DS1 genes will provide sequence information covering the region where the A2 antisense and the B sense primers anneal i.e., around nucleotide 331 in exon 4. The small arrows under the exons denote the positions of sequencing primers that anneal in the introns and that provide the sequence of both sense and antisense DNA strands for each exon. Tables 2 and 3 list the amplification and sequencing primers for all the KIR loci and describe their annealing sites.