University of Minnesota

Jose J Barbosa

Pedro Santamaria

Michael T Boyce-Jacino

Stephen S Rich

Anthony J Faras

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.

DNA sequence-based HLA typing method

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.

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 I HLA genotypes can be unambiguously determined in any subject in 16-24 hours by direct sequencing of HLA-A, HLA-B, and HLA-C transcripts enzymatically amplified and sequenced using a limited number of selected oligonucleotide.

DNA sequence-based HLA class I typing method

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 I HLA genotypes can be unambiguously determined in any subject in 16-24 hours by direct sequencing of HLA-A, HLA-B, and HLA-C transcripts enzymatically amplified and sequenced using a limited number of selected oligonucleotides.

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Roseville, MN, United States of America

Jose J Barbosa