ArticleJournal of immunological methods2013
A method for killer-cell immunoglobulin-like receptor (KIR) 3DL1/3DS1 genotyping using DNA recovered from frozen plasma.
Article in Journal of immunological methods, 2013. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Not yet cited in PubMed.
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6 authors at 2 institutions in 1 country.
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Abstract
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/μl. 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/μl. This method provides reliable and accurate KIR3DL1/S1 typing when conventional sources of high-quality genomic DNA are unavailable or limiting.
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