KIR Receptor Genotyping

Case Number CH-051

Inventors: Elizabeth Trachtenberg, PhD

Patent No. Title Issue Date Country
Pending Methods and Compositions for (KIR) Genotyping   United States

Abstract
The invention provides methods and compositions for single nucleotide polymorphism (SNP)-based killer cell immunoglobulin-like receptor (KIR) gene cluster genotyping using the matrix-assisted laser desorption-ionization time-of-flight (MALDI-TOF) mass spectrometer by amplifying target sequences of KIR genes, and detecting the presence or absence of SNPs of the KIR genes by MALDI-TOF mass spectrometry.

Diversity in terms of both number and combination of KIR genes exists among individuals, as well as extensive allele polymorphism, all of which affect the strength and breadth of the immune response.

Traditional KIR genotyping methods demonstrate limitations overcome by the present invention. Sequence-specific priming (SSP) assay amplifications require that genomic DNA be amplified using a collection of primers in separate reactions in order to define the various loci or alleles to be detected by fragment lengths using gel electrophoresis. This requires many different annealing and extension conditions, which are machine and technologist time intensive, and not conducive to high-throughput analysis. Another limitation of the current SSP method is that it requires a large quantity of high quality DNA (>5g). Further, the SSP method poses the problem of sample amplification failure, which could be due to either general PCR failure or a sequence variant, which could result in erroneous KIR genotyping results.

An alternative assay uses sequence specific oligonucleotide probes (SSOP) developed for the locus-specific resolution of the 14 KIR genes. This requires a small quantity of genomic DNA (50-100ng) amplified at four KIR domains. PCR products are then denatured and vacuum blotted onto replicate 96-sample nylon membranes. Replicate membranes are hybridized to 39 sequence-specific probes, washed under stringent conditions to remove unbound probe, and are then decoded using a computer program. Although generally more efficient than the SSP methods, genotyping analysis by SSOP assay is still cumbersome.

Advantage
The methods of this invention involve amplifying a plurality of target sequences of a plurality of KIR genes and detecting the presence or absence of a plurality of single SNPs of the plurality of KIR genes.

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