P136 The assay for simultaneous genotyping of 14 functional KIR genes by sequence-based typing

Abstract

Aim The killer cell immunoglobulin-like receptors (KIRs) gene cluster consists of 14 functional genes and 2 pseudogenes. Identification of KIR alleles has functional and clinical significance. However, KIR sequencing-based typing (SBT) commercial kit is not available. High through-put simultaneously genotyping of 14 functional KIR genes by SBT has not been reported owing to the varied PCR annealing temperature or the varied fragment size of PCR amplicons and extension time. The aim of present study aimed to establish the through-put KIR SBT method. Methods We designed and synthesized a total of 112 pairs of KIR gene specific PCR primers and 230 sequencing primers for SBT test of 14 functional KIR genes. PCR amplification of all exons of each KIR gene was conducted by using 3–5 pairs KIR gene-specific PCR primers. All the KIR genes were amplified under the same PCR conditions: 95 °C 2 min; followed by 35 cycles of 15 s. at 95 °C, 15 s. at 68 °C, 3.5 min. at 72 °C; 7 min. at 72 °C;4 °C forever. The PCR product was run electrophoresis on the agarose gel to identify the presence or absence of KIR genes using our KIR-SBT PCR primers, as a parallel control, the presence and absence of KIR genes were also examined using the Inno-Train KIR-SSP commercial kit. Samples with the positive specific PCR products were then subjected to sequencing on an ABI 3730 DNA Sequencer. Sequence assembly and analysis were accomplished using the Assign 3.5 software (Conexio Genomics, Australia). Results All the 14 functional KIR genes can be amplified simultaneously under the same PCR condition and the PCR procedure can be finished in less than 3.5 h, which greatly facility the PCR procedure and make it less labour-consuming. The presence or absence of 14 functional KIR genes identified by our KIR-SBT PCR primers were completely in accordance with the results of KIR SSP commercial kit, indicating our KIR-SBT PCR primers could ensure specific amplification for each KIR gene. No background or noise was emerged in the obtained sequences. 306 samples from southern Hans were detected by our KIR SBT method, 115 alleles including 46 KIR novel alleles and 258 allelic KIR profiles were identified. Conclusions The through-put SBT method developed by us shows a broad prospective in transplantation, KIR-associated diseases and population studies.