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NON‐RADIOACTIVE OLIGOTYPING FOR HLA‐DR1‐DRw10 USING POLYMERASE CHAIN REACTION, DIGOXIGENIN‐LABELLED OLIGONUCLEOTIDES AND CHEMILUMINESCENCE DETECTION
Author(s) -
NevinnyStickel C.,
Hinzpeter M.,
Andreas A.,
Albert E.D.
Publication year - 1991
Publication title -
international journal of immunogenetics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.41
H-Index - 47
eISSN - 1744-313X
pISSN - 1744-3121
DOI - 10.1111/j.1744-313x.1991.tb00032.x
Subject(s) - digoxigenin , oligonucleotide , chemiluminescence , polymerase chain reaction , chemistry , microbiology and biotechnology , alkaline phosphatase , dna , biochemistry , biology , chromatography , gene , enzyme , in situ hybridization , messenger rna
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.