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

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.

p53 gene mutations are not required for early dissemination of cancer cells

“…Total RNA was isolated from 10 cell lines recently established from micrometastatic BM tumor cells by immortalization with simian virus 40 (SV40) large T antigen DNA (13). The identities of the cell lines were confirmed by HLA-DRB1* typing using either genomic DNA of cell lines and peripheral blood lymphocytes or DNA isolated from cytospins preparations of the patients, following the oligonucleotide typing system protocol as described by Nevinny-Stickel et al (29). Total RNA was purified to poly(A)-RNA by using the Oligotex mRNA Mini Kit (Quiagen).…”

Early expression and high prevalence of islet autoantibodies for DR3/4 heterozygous and DR4/4 homozygous offspring of parents with Type I diabetes: The German BABYDIAB study

“…For the dot blot hybridization the amplified DNA (2 ml) was spotted onto nylon membranes (Boehringer Mannheim, Mannheim, Germany) and hybridized with 15 DRB generic, 17 DQA and 20 DQB specific oligonucleotides, respectively [14]. The hybridized oligonucleotides 3 ¢ end labelled with digoxigenin-11±2 ¢,3 ¢-dideoxyuridine-5 ¢-triphosphate (Dig-11-ddUTP, Boehringer Mannheim) were detected using anti-dioxigenin-AP Fab fragments (Boehringer Mannheim) and made visible with the chemiluminescent substrate CSPD (Boehringer Mannheim) [15,16]. The HLA-DRB1, HLA-DQA1 and HLA-DQB1 alleles were named according to the nomenclature of 1996 [17].…”

HLA markers and prediction of clinical course and outcome in rheumatoid arthritis

“…Each cycle consisted of denaturation at 94°C for 20 seconds, annealing at 55°C for 20 seconds, and extension at 72°C for 30 seconds. PCR products were dotted on nylon membranes hybridized to sequence-specific oligonucleotides (SSO), as described by Nevinny-Stickel and coworkers (47). For identification of DRB1*02, the above protocol was supplemented by the use of D11 (5'-GCAGCAGGATAA-GTATGAG-3') (48).…”