A method for Killer-cell Immunoglobulin-like Receptor (KIR) 3DL1/3DS1 genotyping using DNA recovered from frozen plasma

作者：Shahid Aniqa; Chopera Denis R; Martin Eric; Penney Kali A; Milloy M J; Brumme Zabrina L^*

来源：Journal of Immunological Methods, 2013, 391(1-2): 154-162.

DOI：10.1016/j.jim.2013.03.005

• Killer-cell immunoglobulin receptor
• KIR3DL1/S1
• Plasma
• Genotyping

We describe a reliable and semi-automated method for Killer-cell Immunoglobulin-like Receptor (KIR) 3DL1/S1 genotyping using DNA recovered from frozen plasma. The primers and protocol were first validated using two independent genomic DNA reference panels. To confirm the approach using plasma-derived DNA, total nucleic acids were extracted from 69 paired frozen PBMC and plasma specimens representing all common KIR3DL1/S1 genotypes (3DS1/3DS1, 3DS1/3DL1 and 3DL1/3DL1, including rare allele 3DL1*054), and analyzed in a blinded fashion. The method involves independent nested PCR amplification of KIR3DL1/S1 Exon 4, and if required Exon 3, using universal sequence-specific primers, followed by bidirectional sequencing. The free basecalling software RECall is recommended for rapid, semi-automated chromatogram analysis. KIR3DL1/S1 type assignment is based on two key nucleotide polymorphisms in Exon 4 and, if required, up to two additional polymorphisms in exon 3. Assignment can be performed manually or using our web-based algorithm, KIR3D. Extractions from plasma yielded median [IQR] nucleic acid concentrations of 0.9 [below the limit of detection-2.45] ng/mu l. PCR was successful for 100% of exon 4 (69/69) and exon 3 (29/29) plasma amplifications. Chromatogram quality was high and concordance between PBMC and plasma-derived types was 100%. The estimated lower limit of input DNA required for reliable typing is 0.01 ng/mu l. This method provides reliable and accurate KIR3DL1/S1 typing when conventional sources of high-quality genomic DNA are unavailable or limiting.

• 出版日期2013-5-31