Enzyme-linked DNA oligotyping. A practical method for clinical HLA-DNA typing.
The use of PCR and oligonucleotide hybridization has increased the accuracy and resolution of typing for HLA class II alleles, but current procedures, performed in batches, take too long and are not suited for testing single samples. We have developed a typing method using enzyme-linked oligonucleotides and PCR products immobilized in 96-well trays. Trays preloaded with typing probes, covalently linked with alkaline phosphatase, have been kept for weeks at 4 degrees C without loss of enzyme-probe activity. Bound alkaline phosphatase was detected using a color reaction with enzymatic amplification which produces readings in 30 minutes. Coupled with a quick DNA preparation method, results can be obtained in about 4 hours. This method can be easily performed in small laboratories. It is accurate, reproducible, and sensitive, and will make oligotyping for HLA alleles more convenient for testing clinical samples.
Lazaro
A M
AM
Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas 75235-8886.
Fernandez-Viña
M A
MA
Liu
Z
Z
Stastny
P
P
D000469:Alkaline Phosphatase; D000483:Alleles; D001483:Base Sequence; D004247:DNA / Q000032:analysis*; D005802:Genes, MHC Class II / Q000235:genetics; D006684:HLA-DR Antigens / Q000235:genetics*; D006650:Histocompatibility Testing / Q000379:methods*; D006801:Humans; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes; D009841:Oligonucleotides / Q000235:genetics*; D016133:Polymerase Chain Reaction
D006684:HLA-DR Antigens; D015345:Oligonucleotide Probes; D009841:Oligonucleotides; D004247:DNA; D000469:Alkaline Phosphatase