Enrichment of individual KIR2DL4 sequences from genomic DNA using long‐template PCR and allele‐specific hybridization to magnetic bead‐bound oligonucleotide probes

Abstract

DNA enrichment by allele-specific hybridization (DEASH) was used as a means to isolate individual alleles of the killer cell immunoglobulin-like receptor (KIR2DL4) gene from heterozygous genomic DNA. Using long-template polymerase chain reaction (LT-PCR), the complete KIR2DL4 gene was amplified from a cell line that had previously been characterized for its KIR gene content by PCR using sequence-specific primers (PCR-SSP). The whole gene amplicons were sequenced and we identified two heterozygous positions in accordance with the predictions of the PCR-SSP. The amplicons were then hybridized to allele-specific, biotinylated oligonucleotide probes and through binding to streptavidin-coated beads, the targeted alleles were enriched. A second PCR amplified only the exonic regions of the enriched allele, and these were then sequenced in full. We show DEASH to be capable of enriching single alleles from a heterozygous PCR product, and through sequencing the enriched DNA, we are able to produce complete coding sequences of the KIR2DL4 alleles in accordance with the typing predicted by PCR-SSP.

This article has been retracted: A gene‐specific primer extension and liquid bead array system for killer cell immunoglobulin‐like receptor genotyping

“…For example, various KIR and MHC class I molecules have been attributed to infection resistance, autoimmune disease, cancer, reproduction, and the facility of hematopoietic stem cell transplantation (3–7). The importance of KIR genotyping has thus increased, and various KIR genotyping technologies have been developed using sequence‐specific oligonucleotide probes (SSOPs) (8), reverse sequence‐specific oligonucleotides (rSSOs) (9), Matrix‐assisted laser desorption/ ionisation‐time of flight (MALDI‐TOF) mass spectrometry (10), magnetic bead‐bound oligonucleotide probes (11), and pyrosequencing (12). Sequence‐specific primer directed polymerase chain reaction (SSP‐PCR) technology has also been applied to identify the presence and absence of the 16 KIR genes (13, 14).…”

KIR3DL2: diversity in a hematopoietic stem cell transplant population

“…The diversity at the 3DL2 locus continues to increase although some alleles appear to be commonly found in the Caucasian population. Development of strategies to link polymorphisms separated by long genetic distances to resolve ambiguous combinations such as that described by Roberts and colleagues (12) is needed. The differences among alleles are relatively modest in number and their impact on 3DL2 function is not yet known.…”

Seventeen novel alleles add to the already extensive KIR3DL3 diversity

“…The captured haploid DNA was purified with a series of wash steps, and the targeted DNA fragments were amplified by PCR using 3DL3 primer pairs adding five additional PCR cycles. A similar strategy was described (7).…”