C - Chemistry – Metallurgy – 12 – Q
Patent
C - Chemistry, Metallurgy
12
Q
C12Q 1/68 (2006.01) C07H 21/04 (2006.01)
Patent
CA 2105585
2105585 9215711 PCTABS00016 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.
Barbosa Jose Joaquim
Boyce-Jacino Michael Thomas
Faras Anthony James
Rich Stephen Saul
Santamaria Pedro
Goudreau Gage Dubuc
The Regents Of The University Of Minnesota
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