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.
Labor für Immungenetik, Kinderpoliklinik, LMU München, Germany.
C
C
Nevinny-Stickel
M D
MD
Bettinotti
A
A
Andreas
M
M
Hinzpeter
K
K
Mühlegger
G
G
Schmitz
E D
ED
Albert
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