title: A method for killer-cell immunoglobulin-like receptor (KIR) 3DL1/3DS1 genotyping using DNA recovered from frozen plasma.
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.
authors.affiliation: Faculty of Health Sciences, Simon Fraser University, Burnaby, British Columbia, Canada.
authors.forename: Aniqa
authors.initials: A
authors.lastname: Shahid
authors.forename: Denis R
authors.initials: DR
authors.lastname: Chopera
authors.forename: Eric
authors.initials: E
authors.lastname: Martin
authors.forename: Kali A
authors.initials: KA
authors.lastname: Penney
authors.forename: M-J
authors.initials: MJ
authors.lastname: Milloy
authors.forename: Zabrina L
authors.initials: ZL
authors.lastname: Brumme
mesh_terms: D000465:Algorithms; D057205:Automation, Laboratory; D004247:DNA / Q000097:blood*; D005091:Exons; D005615:Freezing; D005838:Genotype; D006801:Humans; D057230:Limit of Detection; D016133:Polymerase Chain Reaction / Q000592:standards; D020641:Polymorphism, Single Nucleotide; D054347:Receptors, KIR3DL2 / Q000097:blood / Q000235:genetics*; D054349:Receptors, KIR3DS1 / Q000097:blood / Q000235:genetics*; D012015:Reference Standards; D015203:Reproducibility of Results; D017422:Sequence Analysis, DNA / Q000379:methods* / Q000592:standards; D012985:Software Design; D057188:Workflow
chemical_list: D054347:Receptors, KIR3DL2; D054349:Receptors, KIR3DS1; D004247:DNA