This was a study of detection systems for DQ alpha HLA polymorphism that could be exploited for the demonstration of simulated chimerism. Polymorphic segments of DO alpha DNA were amplified by the polymerase chain reaction (PCR). Simulated chimerism was represented by a mixture of minor and major component DNA. The goal was to detect minor component DNA in the presence of major component DNA utilizing various laboratory techniques. Techniques studied included probe strip typing with the AmpliType HLA-DO Alpha test kit, allele-specific amplification, polyacrylamide gel electrophoresis, restriction enzymes, and Southern transfer combined with a peroxidase detection system.
The AmpliType HLA-DO Alpha test kit had a detection sensitivity of at least 0.2%. This is much better than the 10% detection sensitivity in non-PCR techniques. When the 3.0 DC alpha type was mixed as the minor component with undiluted 1.1 DQ alpha type, the detection sensitivity for the 3.0 DC alpha type increased to a detection level of 0.1%.
The allele-specific primers were able to specifically amplify the minor component DNA in the presence of major component DNA. Major component DNA did not amplify and thus did not compete with the minor component DNA for Taq polymerase. The allele-specific primers provided an overall detection sensitivity of 0.2%.
Background interference prevented detection of minor component bands on both polyacrylamide gels stained with ethidium bromide and on Southern blots reacted with the peroxidase detection system.
| Identifer | oai:union.ndltd.org:vcu.edu/oai:scholarscompass.vcu.edu:etd-6297 |
| Date | 01 January 1991 |
| Creators | Mello, Richard |
| Publisher | VCU Scholars Compass |
| Source Sets | Virginia Commonwealth University |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Theses and Dissertations |
| Rights | © The Author |
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