NON‐RADIOACTIVE OLIGOTYPING FOR HLA‐DR1‐DRw10 USING POLYMERASE CHAIN REACTION, DIGOXIGENIN‐LABELLED OLIGONUCLEOTIDES AND CHEMILUMINESCENCE DETECTION

Abstract

SUMMARY 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 chemilumine‐scent substate 3‐(2‘‐spiroadamantan)‐4‐(3′‐phosphoryloxy)‐phenyl‐1,2‐dioxetan (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.