A method for determining the genotype of one or more HLA Class H polymorphic gene loci of interest selected from the group consisting of HLA-DRB1/3/4/5, --DQB1, --DQA1, --DPB1 and --DPA1, of n subject in a sample containing subject RNA using a combination of conserved and non-conserved oligonucleotide primers, said method comprising:
- (a) synthesizing in separate reactions, from RNA isolated from said sample, a cDNA molecule for each allele of each gene locus of interest, using a conserved oligonucleotide antisense primer that anneals to a nucleotide region conserved between each allele of each gene loci to be genotyped;
- (b) amplifying said cDNA by at least one amplifying reaction to generate sufficient first and second double stranded amplifying reaction products for sequencing, using in a separate first amplifying reaction for each of said gene loci of interest encoded by the 3'-end of the alpha 1 domain exon, an oligonucleotide primer pair comprising a first conserved primer and a second conserved primer or, when said gene locus is HLA-DQB or HLA-DRB, optionally a first nonconserved primer for each of said gene loci of interest, wherein one of said first and second conserved oligonucleotide primers anneals to at least one allele of each of said loci of interest;
- (c) sequencing directly at least one strand of said amplifying reaction product of each of said gene loci of interest to produce at least one sequencing ladder for each of said gene loci of interest, using in each sequencing reaction a third conserved oligonucleotide primer for each of said gene loci of interest;
- (d) comparing the nucleotide sequence obtained from each sequencing ladder to known sequences for each such gene locus;
- and, optionally when said gene locus is HLA-DQB or HLA-DRB,
- (e) comparing the nucleotide sequence of each sequencing ladder optionally obtained with a conserved oligonucleotide primer pair to the nucleotide sequence of each allele of such gene locus amplified with a conserved/nonconserved oligonucleotide primer pair, wherein said gene loci of interest are one or more gene loci whereby the Zenotype of said HLA Class II gene locus is obtained,
- wherein when said gene locus is HLA-DQA1 (a) said conserved oligonucleotide antisense primer is depicted in SEQ. ID. NO:
- 3;
- (b) said first and second conserved primers are depicted in SEQ. ID. NO:
- 3 and SEQ. ID. NO:
- 7:
- and (c) said third conserved primer is depicted in SEQ. ID. NO:
- 14 or SEQ. ID. NO:
- 15;
- wherein when said gene locus is HLA:DOB1 said combination of conserved and non-conserved oligonucleotide primers is selected from the group consisting of
- (1) (a) said conserved oligonucleotide antisense primer is depicted in SEQ. ID. NO:
- 1:
- (b) said first and second conserved primers are depicted in SEQ. ID. NO:
- 1 and SEQ. ID. NO:
- 4;
- and (c) said third conserved primer is depicted in SEQ. ID. NO:
- 10 or SEQ. ID. NO:
- 12;
- (2) (a) said conserved oligonucleotide antisense primer is depicted in SEQ. ID. NO:
- 1:
- (b) said tint conserved primer is depicted in SEQ. ID. NO:
- 1 and said first nonconserved primer is depicted SEQ. ID. NO:
- 47 and wherein annealing of said conserved/nonconserved primer pair occurs at about 37.degree. C.;
- and (c) said third conserved primer is depicted in SEQ. ID. NO:
- 4;
- (3) (a) said conserved oligonucleotide antisense primer is depicted in SEQ. ID. NO:
- 1:
- (b) said first conserved primer is depicted in SEQ. ID. NO:
- 1 and said first nonconserved primer is depicted SEQ. ID. NO:
- 47 and wherein annealing of said conserved/nonconserved primer pair occurs at about 55.degree. C.-60.degree. C.;
- and (c) said third conserved primer is depicted in SEQ. ID. NO:
- 10or SEQ. ID. NO:
- 12;
- (4) (a) said conserved oligonucleotide antisense primer is depicted in SEQ. ID. NO:
- 1:
- (b) said first conserved primer is depicted in SEQ. ID. NO:
- 1 and said first nonconserved primer is depicted SEQ. ID. NO:
- 48;
- and (c) said third conserved primer is depicted in SEQ. ID. NO:
- 10 or SEQ. ID. NO:
- 12;
- and
- (5) (a) said conserved oligonucleotide antisense primer is depicted in SEQ. ID. NO:
- 1:
- (b) said first conserved primer is depicted in SEQ. ID. NO:
- 1 and said first nonconserved primer is depicted SEQ. ID. NO:
- 49:
- and (c) said third conserved primer is depicted in SEQ. ID. NO:
- 10 or SEQ. ID. NO:
- 12;
- wherein when said gene locus is HLA-DPB1, said combination of conserved and non-conserved oligonucleotide primers is selected from the group consisting of
- (1) (a) said conserved oligonucleotide antisense primer is depicted in SEQ. ID. NO:
- 24;
- (b) said first and second conserved primers are depicted in SEQ. ID. NO:
- 24 and SEQ. ID. NO:
- 23:
- and (c) said third conserved primer is depicted in SEQ. ID. NO:
- 25 or SEQ. ID. NO:
- 26:
- and
- (2) (a) said conserved oligonucleotide antisense primer is depicted in SEQ. ID. NO:
- 25;
- (b) said first and second conserved primers are depicted in SEQ. ID. NO:
- 25 and SEQ. ID. NO:
- 23:
- and (c) said third conserved primer is depicted in SEQ. ID. NO:
- 26;
- wherein when said gene locus is HLA-DPA1, said combination of conserved and non-conserved oligonucleotide primers is selected from the group consisting of
- (1) (a) said conserved oligonucleotide antisense primer is depicted in SEQ. ID. NO:
- 31;
- (b) said first and second conserved primers are depicted in SEQ. ID. NO:
- 31 and SEQ. ID. NO:
- 32;
- and (c) said third conserved primer is depicted in SEQ. ID. NO:
- 33 or SEQ. ID. NO:
- 34;
- (2) (a) said conserved oligonucleotide and sense primer is depicted in SEQ. ID. NO:
- 39;
- (b) said first and second conserved primers are depicted in SEQ. ID. NO:
- 39 and SEQ. ID. NO:
- 38:
- and (c) said third conserved primer is depicted in SEQ. ID. NO:
- 40 or SEQ. ID. NO:
- 41;
- (3) (a) said conserved oligonucleotide antisense primer is depicted in SEQ. ID. NO:
- 33;
- (b) said first and second conserved primers are depicted in SEQ. ID. NO:
- 33 and SEQ. ID. NO:
- 32;
- and (c) said third conserved primer is depicted in SEQ. ID. NO:
- 34;
- wherein when said gene locus is HLA-DRB, said combination of conserved and non-conserved oligonucleotide primers is selected from the group consisting of
- (1) (a) said conserved oligonucleotide and sense primer is depicted in SEQ. ID. NO:
- 2:
- (b) said first and second conserved primers are depicted in SEQ. ID. NO:
- 2 and SEQ. ID. NO:
- 6:
- and (c) said third conserved primer is depicted in SEQ. ID. NO:
- 11 or SEQ. ID. NO:
- 13;
- (2) (a) said conserved oligonucleotide antisense primer is depicted in SEQ. ID. NO:
- 2;
- (b) said first conserved primer is depicted in SEQ. ID. NO:
- 2 and said first nonconserved primer is depicted in SEQ. ID, NO:
- 42 and (c) said third conserved primer is depicted in SEQ. ID. NO:
- 11 or SEQ. ID. NO:
- 13;
- (3) (a) said conserved oligonucleotide antisense primer is depicted in SEQ. ID. NO:
- 2;
- (b) said first conserved primer is depicted in SEQ. ID. NO:
- 2 and said first nonconserved primer is depicted in SEQ, ID. NO:
- 43;
- and (c) said third conserved primer is depicted in SEQ. ID. NO:
- 11 or SEQ, ID. NO:
- 13;
- (4) (a) said conserved oligonucleotide antisense primer is depicted in SEQ. ID. NO:
- 2;
- (b) said first conserved primer is depicted in SEQ, ID. NO:
- 2 and said first nonconserved primer is depicted in SEQ. ID, NO:
- 44:
- and (c) said third conserved primer is depicted in SEQ. ID. NO:
- 11 or SEQ. ID. NO:
- 13;
- (5) (a) said conserved oligonucleotide antisense primer is depicted in SEQ. ID. NO:
- 2;
- (b) said amplifying has four discrete amplifying steps, and wherein in first discrete amplifying step said first conserved primer is depicted in SEQ. ID. NO:
- 2 and said second conserved primer is depicted in SEQ. ID. NO:
- 6:
- and in a second discrete amplifying step said first conserved primer is depicted in SEQ. ID. NO:
- 2 and said first nonconserved primer is depicted in SEQ. ID. NO:
- 42), and in a third discrete amplifying step said first conserved primer is depicted in SEQ. ID. NO:
- 2 and said second nonconserved primer is depicted in SEQ. ID. NO:
- 43 and in a fourth discrete amplifying step said first conserved primer is depicted in SEQ. ID. NO:
- 2 and said third nonconserved primer is depicted in SEQ. ID. NO:
- 44;
- and (c) said third conserved primer is depicted in SEQ. ID. NO:
- 11 or SEQ. ID. NO:
- 13 or SEQ. ID. NO:
- 5;
- (6) (a) said conserved oligonucleotide antisense primer is depicted in SEQ. ID. NO:
- 2;
- (b) said first conserved primer is depicted in SEQ. ID. NO:
- 2 and said first nonconserved primer is depicted in SEQ. ID. NO:
- 45;
- and (c) said third conserved primer is depicted in SEQ. ID. NO:
- 11 or SEQ. ID. NO:
- 13;
- (7) (a) said conserved oligonucleotide antisense primer is depicted in SEQ. ID. NO:
- 2;
- (b) said first conserved primer is depicted in SEQ. ID. NO:
- 2 and said first nonconserved primer is depicted in SEQ. ID. NO:
- 46:
- and (c) said third conserved primer is depicted in SEQ. ID. NO:
- 11 or SEQ. ID. NO:
- 13;
- and
- (8) (a) said conserved oligonucleotide antisense primer is depicted in SEQ. ID. NO:
- 2;
- (b) said first and second conserved primers are depicted in SEQ. ID. NO:
- 2 and SEQ. ID. NO:
- 5;
- and (c) said third conserved primer is depicted in SEQ. ID. NO:
- 11 or SEQ. ID. NO:
- 13.