| Popis: |
The limiting detection signal for identification of human genetic markers, such as HLA-D and VNTR genes, was determined using DNA isolated from a series of decreasing numbers of lymphocytes carrying the target marker in the polymerase chain reaction (PCR). The PCR procedure was assembled by incorporating 32P-labeled dCTP in the reaction mixture. Primers specific for detection of MHC Class II genes such as HLA-DR1, -DR2, -DRw52 and -DRw53 were utilized when cells were mismatched by one DR type, and primers for the identification of the region of variable number of tandem repeats (VNTRs) were utilized where cells had the same DR types. The 32P-incorporated amplified DNA was analyzed by polyacrylamide gel electrophoresis followed by exposure to x-ray film. The sensitivity of the test varied for different allelic markers as evaluated by amplification of DNA from each set of a mixture of lymphocytes. The target HLA-DR markers were detectable in a cell ratio of as high as 1:100,000, whereas the VNTR markers were detectable at a 1:1000 cell ratio. The approach described here offers certain advantages: 1) increased sensitivity, 2) quantitative power, 3) reduced assay time, 4) simplified procedure and 5) less expense. This method provides valuable information for studies involving forensic specimens and marrow engraftment after allogenic bone marrow transplantation (BMT) that require discrete representation of one allele relative to another in a heterozygous sample where limited quantities of target DNA are available. |
