Enrichment of individual KIR2DL4 sequences from genomic DNA using long-template PCR and allele-specific hybridization to magnetic bead-bound oligonucleotide probes.
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.
Roberts
C H
CH
Anthony Nolan Research Institute, The Royal Free Hospital, London, UK.
Turino
C
C
Madrigal
J A
JA
Marsh
S G E
SG
D000483:Alleles; D001483:Base Sequence; D004247:DNA / Q000235:genetics* / Q000302:isolation & purification*; D006801:Humans; D008280:Magnetics; D009693:Nucleic Acid Hybridization / Q000379:methods; D015345:Oligonucleotide Probes / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods; D011971:Receptors, Immunologic / Q000235:genetics*; D054340:Receptors, KIR; D054345:Receptors, KIR2DL4
C515079:KIR2DL4 protein, human; D015345:Oligonucleotide Probes; D011971:Receptors, Immunologic; D054340:Receptors, KIR; D054345:Receptors, KIR2DL4; D004247:DNA