Cost-effective and fast KIR gene-content genotyping by multiplex melting curve analysis.
Killer cell immunoglobulin-like receptor (KIR) genes encode cell surface molecules that recognize HLA molecules and modulate the activity of natural killer (NK) cells. KIR genes exhibit presence and absence polymorphism, which generates a variety of gene-content haplotypes in worldwide populations. KIR gene-content variation is implicated in many diseases and is also important for placentation and transplantation. Because of the complexity of KIR polymorphism, variation in this family is still mostly studied at the gene-content level, even with the advent of next-generation sequencing (NGS) methods. Gene-content determination is generally expensive and/or time-consuming. To overcome these difficulties, we developed a method based on multiplex polymerase chain reaction with specific sequence primers (PCR-SSP) followed by melting curve analysis that allows cost-effective, precise and fast generation of results. Our method was 100% concordant with a gel-based method and 99.9% concordant with presence and absence determination by NGS. The limit of detection for accurate typing was 30 ng of DNA (0.42 μM) with 260/230 and 260/280 ratios as low as 0.19 and of 0.44. In addition, we developed a user-friendly Java-based computational application called killerPeak that interprets the raw data generated by Viia7 or QuantStudio 7 quantitative PCR machines and reliably exports the final genotyping results in spreadsheet file format. The combination of a reliable method that requires low amount of DNA with an automated interpretation of results allows scaling the KIR genotyping in large cohorts with reduced turnaround time.
['Amorim|Leonardo M|LM|', 'Santos|Tiago H S|THS|', 'Hollenbach|Jill A|JA|', 'Norman|Paul J|PJ|', 'Marin|Wesley M|WM|', 'Dandekar|Ravi|R|', 'Ribeiro|Enilze M S F|EMSF|', 'Petzl-Erler|Maria L|ML|', 'Augusto|Danillo G|DG|0000-0001-5665-8547']
D017931:DNA Primers D015870:Gene Expression D005838:Genotype D060005:Genotyping Techniques D059014:High-Throughput Nucleotide Sequencing D006801:Humans D007694:Killer Cells, Natural D057230:Limit of Detection D060885:Multiplex Polymerase Chain Reaction D009691:Nucleic Acid Denaturation D011110:Polymorphism, Genetic D054340:Receptors, KIR D012984:Software
genotyping PCR-SSP killer-cell immunoglobulin-like receptors qPCR real-time