Design and validation of a multiplex specific primer‐directed polymerase chain reaction assay for killer‐cell immunoglobulin‐like receptor genetic profiling

Abstract

Current methodologies for the analysis of the killer-cell immunoglobulin-like receptor (KIR) locus utilize specific primer-directed polymerase chain reaction (SSP-PCR), which require a wide range of DNA input, multiple reaction conditions, and up to 16 individual reactions. We have developed and validated a multiplex SSP-PCR method for the genetic analysis of the KIR locus. Design and optimization of four multiplex groups targeting 14 genes and their alleles on the KIR locus has been completed. Each multiplex group contains PCR products that differ in size by a minimum of 15 bp to allow sufficient fragment length resolution for size discrimination by gel electrophoresis. This assay allows for efficient genotyping of the KIR locus while requiring a minimum amount of DNA input, utilizing the simplicity of SSP-PCR.

Killer cell immunoglobulin like receptor gene association with tuberculosis

“…KIR genotyping was performed using multiplex Sequence-Specific Primer directed Polymerase Chain reaction (SSP-PCR). SSP-PCR designing and Primer sequences for 14 KIR genes KIR2DL1, KIR2DS4, KIR2DL3, KIR2DL5 (as there was no difference between KIR2DL5A andKIR2DL5B), KIR2DL2, KIR2DS2, KIR2DS5, KIR3DS1, KIR3DL1, KIR2DS3, KIR2DS1, KIR3DL3, KIR2DL4 and KIR3DL2 (except KIR2DP1 and KIR3DP1 pseudo genes) were adopted from Abalos et al (2010) [23].…”

Methods for Multiplex Template Sampling in Digital PCR Assays

“…We note that the speed and simplicity of dPCR comes at the cost of much less information content as compared to sequencing. Although simultaneous non-quantitative amplification of multiple genomic loci using multiplexed PCR is well-established for sequencing or genotyping purposes [17], [18], [19], and most recently duplexing of dPCR was shown to increase precision [20], the use of high-level multiplexing of dPCR for accurate quantification has not been previously reported. Although we have demonstrated here the 10-fold multiplexing of loci on a single chromosome, accurate determination of allelic ratios in clinical samples would require extension of this method for multiplexed sampling of a reference chromosome, or the multiplexed targeting of multiple loci on different chromosomes.…”

Donor KIR2DS1 reduces the risk of transplant related mortality in HLA-C2 positive young recipients with hematological malignancies treated by myeloablative conditioning

“…HLA-C*03, *07, *12, *15, *16) which were resolved by high resolution typing. Genotyping for the presence of KIR genes was performed by an allele specific multiplex PCR using archived DNA samples of HSCT donors [24]. Briefly, amplifications of 14 genes/variants with 31 primer pairs (oligonucleotide sequences were identical to those of Abalos et al and are available upon request) were performed in 4 multiplex PCR reactions, followed by size separation on agarose gel-electrophoresis.…”