NON‐RADIOACTIVE OLIGOTYPING FOR HLA‐DR1‐DRw10 USING POLYMERASE CHAIN REACTION, DIGOXIGENIN‐LABELLED OLIGONUCLEOTIDES AND CHEMILUMINESCENCE DETECTION
- 1 October 1991
- journal article
- Published by Wiley in International Journal of Immunogenetics
- Vol. 18 (5-6) , 323-332
- https://doi.org/10.1111/j.1744-313x.1991.tb00032.x
Abstract
We describe a rapid non-radioactive DNA typing of the serological types DR1-DRw10 using polymerase chain reaction (PCR)-amplified DNA and 15 sequence-specific oligonucleotides (SSO) which are labelled enzymatically at their 3' end with one digoxigenin (DIG). The hybridized SSOs were detected using anti-DIG alkaline phosphatase and Fab fragments and visualization was obtained with the chemiluminescent substate 3-(2'-spiroadamantan)-4-(3''-phosphoryloxy)-phenyl-1,2-di o xetan (AMPPD). The results were identical with those of the previously used 5-bromo-4-chloro-3-indolyl-phosphate (BCIP)/4-nitrobluetetrazolium chloride (NBT) system. The use of AMPPD is more rapid and allows the repeated rehybridization of the membrane-bound DNA.Keywords
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