alexa Non-radioactive oligotyping for HLA-DR1-DRw10 using polymerase chain reaction, digoxigenin-labelled oligonucleotides and chemiluminescence detection.


Journal of Transplantation Technologies & Research

Author(s): NevinnyStickel C, Hinzpeter M, Andreas A, Albert ED

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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.
This article was published in Eur J Immunogenet and referenced in Journal of Transplantation Technologies & Research

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