#28-OR
EVALUATION OF A SIMPLE PROTOCOL FOR MOLECULAR TYPING OF KIR LOCI USING A SINGLE MULTIPLEX AMPLIFICATION REACTION FOLLOWED BY HYBRIDIZATION TO ALKALINE PHOSPHATASE LABELED PROBES.
Ivan Balazs, Ph.D., Chady Hakim, B.S. and Susannah Kehl, B.S.. Stamford CT, USA, Tepnel Lifecodes Corporation, 10804, R
D.
One of the challenges of performing molecular typing of KIR is the extensive sequence similarity of the 16 loci. The protocols most frequently utilized for KIR typing consist in the amplification of individual loci by SSP and visualization by gel electrophoresis or the amplification of multiple subfamilies of KIR loci followed by SSOP hybridization to digoxigenin probes. To further simplify the typing of large number of samples for prospective or retrospective clinical studies, we evaluated the use of a single multiplex PCR reaction to amplify several exons for all loci. A primer mixture was used to amplify exons 4 and 5 for all loci and exons 8 for 2DL3. A total of 262 DNA samples were amplified either in 3 amplification reactions or in a single multiplex reaction. The amplified DNA was spotted on replicate nylon membranes and hybridized to 17 alkaline phosphatase labeled probes, specific to each of the 16 KIR loci plus 2DS4*003, and the results were visualized by chemiluminescence. The results of both types of amplification generated identical results. As an independent confirmation of these typings, the samples were also typed using an SSP method by an different laboratory. The results obtained by SSOP and SSP methods show a correlation >99.6% indicating that, with few exceptions, both procedures generated the same results. The few discrepancies are being further analyzed to determine whether they represent typing errors or new alleles detected by the SSOP or SSP methods. In conclusion, this SSOP method provides a simple protocol for the molecular typing of KIR loci for large number of samples.