Facilitation of KIR genotyping by a PCR-SSP method that amplifies short DNA fragments.
Detection of killer-cell immunoglobulin-like receptors (KIR) genes by polymerase chain reaction with sequence-specific primers (PCR-SSP) led in 1997 to the discovery that human genomes diverge largely in the KIR they encode. While only a few KIR genes are conserved in all humans, most individuals lack several those genes, which tend to associate in diverse haplotypic combinations. The PCR-SSP technique, updated to detect the more recently identified KIR genes and alleles, is still used widely to analyze the diversity of human populations, and to study the influence of KIR-gene variability on human health. Several published PCR-SSP methods for KIR genotyping, although simple and robust, have the drawback of relying on the amplification of DNA fragments spanning 0.5-2.0 kbp, which tends to fail in low-quality DNAs. Valuable collections of DNAs often include such poor quality samples, which lead to loss of data and resources. Even worse, undetected falsely negative or positive reactions may result in erroneous gene frequencies and in odd gene combinations. To address those problems, we have redesigned our previously published KIR genotyping method so that it produces short amplicons (less than 200 bp for most genes). This modification minimizes amplification failures, thus conferring greater consistency and reliability to KIR genotyping. In addition, the new PCR-SSP method detects recently described alleles of several KIR genes, and allows for discrimination between the major structural variants of KIR2DS4 and KIR3DP1 without increasing the number of reactions.
Vilches
C
C
Department of Immunology, Hospital Universitario Puerta de Hierro, Madrid, Spain. carlos.vilches@yahoo.com
Castaño
J
J
Gómez-Lozano
N
N
Estefanía
E
E
D005838:Genotype; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods; D018807:Polymorphism, Single-Stranded Conformational; D054340:Receptors, KIR / Q000235:genetics*; D015203:Reproducibility of Results
C473953:KIR2DS4 protein, human; D054340:Receptors, KIR