Nonradioactive HLA class II typing using polymerase chain reaction and digoxigenin-11-2'-3'-dideoxy-uridinetriphosphate-labeled oligonucleotide probes.
We describe a new, simple, rapid, and sensitive nonradioactive technique for the analysis of genetic variations. Genomic DNA was amplified using polymerase chain reaction and amplified DNA was hybridized, with digoxigenin (DIG)-labeled sequence-specific oligonucleotides. High specificity and sensitivity was achieved when labeling the sequence-specific oligonucleotide at the 3' end with only one DIG using digoxigenin-11-2',3'-dideoxy-uridine-5'-triphosphate and DNA deoxynucleotidylexotransferase. The hybridized probes were detected using antidigoxigenin alkaline phosphatase, fab fragments, and X-phosphate/NBT for visualization. This method was applied to the analysis of HLA-DR4-DRB1 alleles in polymerase chain reaction-amplified genomic DNA and resulted in highly specific and sensitive hybridization signals discriminating even in cases of a one-base-pair mismatch. This technique is particularly suited for HLA oligotyping because it allows the use of tetramethylammonium chloride for the simplification of hybridization and washing conditions.
Nevinny-Stickel
C
C
Labor für Immungenetik, Kinderpoliklinik, LMU München, Germany.
Bettinotti
M D
MD
Andreas
A
A
Hinzpeter
M
M
Mühlegger
K
K
Schmitz
G
G
Albert
E D
ED
D001483:Base Sequence; D002460:Cell Line; D003856:Deoxyuracil Nucleotides / Q000235:genetics*; D054306:Dideoxynucleotides; D004076:Digoxigenin / Q000031:analogs & derivatives*; D000949:Histocompatibility Antigens Class II / Q000235:genetics*; D006801:Humans; D016130:Immunophenotyping / Q000379:methods*; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction; D012150:Polymorphism, Restriction Fragment Length
D003856:Deoxyuracil Nucleotides; D054306:Dideoxynucleotides; D000949:Histocompatibility Antigens Class II; D015345:Oligonucleotide Probes; C070581:digoxigenin-11-2',3'-dideoxyuridine 5'-triphosphate; D004076:Digoxigenin