TY - JOUR
T1 - Enzyme-linked DNA oligotyping. A practical method for clinical HLA-DNA typing
AU - Lazaro, Ana M.
AU - Fernandez-Viña, Marcelo A.
AU - Liu, Ziying
AU - Stastny, Peter
N1 - Funding Information:
ACKNOWLEDGMENTS This work was supported in part by NIH grants P01-AI-2327 and R01-HL-47145 and the Texas Advanced Technology Program (grant 003660086). The excellent secretarial help of Glenda Gray is gratefully acknowledged.
PY - 1993/4
Y1 - 1993/4
N2 - 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°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, reproductible, and sensitive, and will make oligotyping for HLA alleles more convenient for testing clinical samples.
AB - 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°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, reproductible, and sensitive, and will make oligotyping for HLA alleles more convenient for testing clinical samples.
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U2 - 10.1016/0198-8859(93)90131-J
DO - 10.1016/0198-8859(93)90131-J
M3 - Article
C2 - 8340233
AN - SCOPUS:0027174823
SN - 0198-8859
VL - 36
SP - 243
EP - 248
JO - Human Immunology
JF - Human Immunology
IS - 4
ER -