Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Patent
1991-03-06
1996-11-26
Wax, Robert A.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
435 15, 435 911, 435 912, 435 9121, 435 915, 536 231, 536 243, 536 2433, 935 8, 935 17, 935 18, 935 77, 935 78, C12Q 148, C12Q 168, C12P 1934, C12N 1511
Patent
active
055784437
ABSTRACT:
The present invention provides a process for determining genotypes in highly polymorphic systems by polymerase chain reaction amplification of cDNA or genomic DNA and direct sequencing polymerase chain reaction products using oligonucleotide primers. More specifically, Class II and Class I HLA genotypes can be unambiguously determined in any subject in 16-24 hours by direct sequencing of DRB, DQB, DQA, DPB, DPA, HLA-A, HLA-B and HLA-C- transcripts enzymatically amplified using a limited number of non-allele-specific oligonucleotides. Total cellular RNA from peripheral blood mononuclear cells is reverse transcribed using antisense primers, specific for different locus (DQB, DQA, DPA or DPB) or group of loci (DRB1-5, or HLA-A and HLA-B and HLA-C). The synthesized cDNA molecules are then enzymatically amplified using different combinations of oligonucleotides for each locus and directly sequenced with Taq polymerase using an internal oligonucleotide. The sequenced genes are then analyzed.
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Barbosa Jose J.
Boyce-Jacino Michael T.
Faras Anthony J.
Rich Stephen S.
Santamaria Pedro
Prouty Rebecca
Regents of the University of Minnesota
Wax Robert A.
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