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HLA CLASS II TYPING IN A MICROTITRE PLATE FORMATE USING DIGOXIGENIN‐LABELLED AMPLIFIED DNA AND BIOTIN‐LABELLED OLIGONUCLEOTIDE PROBES
Author(s) -
NevinnyStickel C.,
Albert E. D.
Publication year - 1993
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.1993.tb00161.x
Subject(s) - digoxigenin , microbiology and biotechnology , oligonucleotide , biotin , hybridization probe , dna , streptavidin , chemistry , dig , genomic dna , biology , biochemistry , in situ hybridization , gene , gene expression
SUMMARY We describe a new, non‐radioactive microtitre plate assay for the analysis of genetic variations at the DNA level. The new method combines hybridization of oligonucleotides with PCR amplified DNA in liquid phase with detection in solid phase using an elisa ‐reader. Genomic DNA is labelled with digoxigenin during PCR using a nucleotide mix containing DIG‐11‐2′‐deoxy‐uridine‐5′‐triphosphate (DIG‐11‐dUTP) DIG labelled, amplified genomic DNA is hybridized in solution with an oligon de which is labelled with one biotin at its 3′‐end, using biotin‐16,2′,3′‐dideoxy ine‐5′‐triphosphate (BIO‐16‐ddUTP) and DNA deoxynucleotidylexo‐transferase (TdT). The hybridized complex is immobilized in a streptavidin (SA) coated microtitre plate via the biotin and detection of digoxigenin is performed using anti‐digoxigenin horseradish peroxidase, fab fragments (anti‐DIG‐POD), and the colorimetric substrate 2,2′‐Azino‐di‐(3‐ethylbenzthiazolinsulfonat[6]) (ABTS®). The resulting absorbtion of the assay is analysed in a microtitre plate reader. This method results in highly specific and sensitive hybridization signals and with the 15 oligonucleotides chosen, allows the typing of DR1‐DR10.