US 10,704,095 B2 · Method and kit for DNA typing of HLA gene

Abstract

The purpose of the present invention is to provide a method and kit for highly precise DNA typing, in which ambiguity derived from phase ambiguity is eliminated. The present invention provides a method for the DNA typing of HLA, which is characterized by comprising: (1) a step of preparing a set of primers which can respectively anneal specifically to an upstream region and a downstream region of each of HLA-A, HLA-B, HLA-C, HLA-DQA1, HLA-DQB1, HLA-DPA1 and HLA-DPB1 gene in the nucleotide sequence for the human genome, and a set of primers which can respectively anneal specifically to exon-2 and a 3′-side non-translated region in HLA-DRB1; (2) a step of carrying out the PCR amplification of a sample to be tested (DNA) using the sets of primers; (3) a step of determining the nucleotide sequence for a PCR-amplified product; and (4) an optional step of carrying out the homology search in a data base.

A method for DNA typing of HLA, comprising the following steps:

(1) a step of preparing a set of primers comprising:
(a) at least one primer subset selected from the group consisting of:
(a1) an (a1) primer subset that can amplify from an enhancer-promoter to a 3′UTR region of HLA-A gene, the (a1) primer subset comprising an (a1) forward primer and an (a1) reverse primer, the (a1) forward primer comprising SEQ ID No. 1 or 2, and the (a1) reverse primer comprising SEQ ID No. 3;
(a2) an (a2) primer subset that can amplify from an enhancer-promoter to a 3′UTR region of HLA-B gene, the (a2) primer subset comprising an (a2) forward primer and an (a2) reverse primer, the (a2) forward primer comprising SEQ ID No. 4, and the (a2) reverse primer comprising SEQ ID No. 5;
(a3) an (a3) primer subset that can amplify from an enhancer-promoter to a 3′UTR region of HLA-C gene, the (a3) primer subset comprising an (a3) forward primer and an (a3) reverse primer, the (a3) forward primer comprising SEQ ID No. 6 or 7, and the (a3) reverse primer comprising SEQ ID No. 8;
(a4) an (a4) primer subset that can amplify from an enhancer-promoter to a 3′UTR region of HLA-DQA1 gene, the (a4) primer subset comprising an (a4) forward primer and an (a4) reverse primer, the (a4) forward primer comprising SEQ ID No. 27 or 46, and the (a4) reverse primer comprising SEQ ID No. 28 or 47;
(a5) an (a5) primer subset that can amplify from an enhancer-promoter to a 3′UTR region of HLA-DQB1 gene, the (a5) primer subset comprising an (a5) forward primer and an (a5) reverse primer, the (a5) forward primer comprising SEQ ID No. 29 or 48, and the (a5) reverse primer comprising SEQ ID No. 30, 49, or 50;
(a6) an (a6) primer subset that can amplify from an enhancer-promoter to a 3′UTR region of HLA-DPA1 gene, the (a6) primer subset comprising an (a6) forward primer and an (a6) reverse primer, the (a6) forward primer comprising SEQ ID No. 23 or 40, and the (a6) reverse primer comprising SEQ ID No. 24 or 41;
and
(a7) an (a7) primer subset that can amplify from an intron or an enhancer-promoter to a 3′UTR region of HLA-DPB1 gene, the (a7) primer subset comprising an (a7) forward primer and an (a7) reverse primer, the (a7) forward primer comprising SEQ ID No. 25, 42, or 44, and the (a7) reverse primer comprising SEQ ID No. 26, 43, or 45;
and
(b) a (b) primer subset that can amplify from exon 2 to a 3′UTR region of HLA-DRB1 gene;
wherein, the (b) primer subset comprises:
(i) a (b1) forward primer comprising SEQ ID No. 9, 10 or 31 and a (b1) reverse primer comprising SEQ ID No. 11 or 32;
(ii) a (b2) forward primer comprising SEQ ID No. 12 or 31 and a (b2) reverse primer comprising SEQ ID No. 11 or 33;
(iii) a (b3) forward primer comprising SEQ ID No. 13 or 34 and a (b3) reverse primer comprising SEQ ID No. 14 or 32;
(iv) a (b4) forward primer comprising SEQ ID No. 15 or 31 and a 04) reverse primer comprising SEQ ID No. 16 or 32;
(v) a (b5) forward primer comprising SEQ ID No. 13 or 31 and a (b5) reverse primer comprising SEQ ID No. 14, 35 or 36;
(vi) a (b6) forward primer comprising SEQ ID No. 13 or 31 and a (b6) reverse primer comprising SEQ ID No. 14, 32 or 37;
(vii) a (b7) forward primer comprising SEQ ID No. 17 or 38 and a (b7) reverse primer comprising SEQ ID No. 18 or 36;
(viii) a (b8) forward primer comprising SEQ ID No. 13 or 31 and a (b8) reverse primer comprising SEQ ID No. 14 or 39;
(ix) a (b9) forward primer comprising SEQ ID No. 19 or 38 and a (b9) reverse primer comprising SEQ ID No. 20 or 36;
and
(x) a (b10) forward primer comprising SEQ ID No. 21 or 31 and a (b10) reverse primer comprising SEQ ID No. 22 or 32;
(2) a step of amplifying a test sample (DNA) by a PCR using the set of primers;
(3) a step of determining the nucleotide sequences of PCR amplified products;
and
(4) a step of optionally carrying out a homology search within a database.

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Method and kit for DNA typing of HLA gene

Life of this patent

Method and kit for DNA typing of HLA gene

Life of this patent

Abstract

Claims18