Abstract
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.
Original language | English (US) |
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Pages (from-to) | 7-13 |
Number of pages | 7 |
Journal | Human Immunology |
Volume | 31 |
Issue number | 1 |
DOIs | |
State | Published - May 1991 |
Externally published | Yes |
ASJC Scopus subject areas
- Immunology and Allergy
- Immunology