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.

1 . A method for DNA typing of HLA, comprising the following steps:
 (1) a step of preparing at least one set of primers selected from the group consisting of primers which 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 genes in human genome sequence, and primers which respectively anneal specifically to exon 2 and a 3′ untranslated region of HLA-DRB1;   (2) a step of amplifying a test sample (DNA) by a PCR using the sets 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.   
     
     
         2 . The method according to  claim 1 , wherein the gene is a HLA-A gene and the sets of primers are selected from oligonucleotides having nucleotide sequences represented by SEQ ID Nos. 1, 2 and 3. 
     
     
         3 . The method according to  claim 1 , wherein the gene is a HLA-B gene, the sets of primers are oligonucleotides having nucleotide sequences represented by SEQ ID Nos. 4 and 5. 
     
     
         4 . The method according to  claim 1 , wherein the gene is a HLA-C gene and the sets of primers are selected from oligonucleotides having nucleotide sequences represented by SEQ ID Nos. 6, 7 and 8. 
     
     
         5 . The method according to  claim 1 , wherein the gene is DR1 type of a HLA-DRB1 gene and the sets of primers are selected from oligonucleotides having nucleotide sequences represented by SEQ ID Nos. 9, 10, 11, 31 and 32. 
     
     
         6 . The method according to  claim 1 , wherein the gene is DR2 type of a HLA-DRB1 gene and the sets of primers are selected from oligonucleotides having nucleotide sequences represented by SEQ ID Nos. 11, 12, 31 and 33. 
     
     
         7 . The method according to  claim 1 , wherein the gene is DR3 type of a HLA-DRB1 gene and the sets of primers are selected from oligonucleotides having nucleotide sequences represented by SEQ ID Nos. 13, 14, 32 and 34. 
     
     
         8 . The method according to  claim 1 , wherein the gene is DR4 type of a HLA-DRB1 gene and the sets of primers are selected from oligonucleotides having nucleotide sequences represented by SEQ ID Nos. 15, 16, 31 and 32. 
     
     
         9 . The method according to  claim 1 , wherein the gene is DR5 type of a HLA-DRB1 gene and the sets of primers are selected from oligonucleotides having nucleotide sequences represented by SEQ ID Nos. 13, 14, 31, 35 and 36. 
     
     
         10 . The method according to  claim 1 , wherein the gene is DR6 type of a HLA-DRB1 gene and the sets of primers are selected from oligonucleotides having nucleotide sequences represented by SEQ ID Nos. 13, 14, 31, 32 and 37. 
     
     
         11 . The method according to  claim 1 , wherein the gene is DR7 type of a HLA-DRB1 gene and the sets of primers are selected from oligonucleotides having nucleotide sequences represented by SEQ ID Nos. 17, 18, 36 and 38. 
     
     
         12 . The method according to  claim 1 , wherein the gene is DR8 type of a HLA-DRB1 gene and the sets of primers are selected from oligonucleotides having nucleotide sequences represented by SEQ ID Nos. 13, 14, 31 and 39. 
     
     
         13 . The method according to  claim 1 , wherein the gene is DR9 type of a HLA-DRB1 gene and the sets of primers are selected from oligonucleotides having nucleotide sequences represented by SEQ ID Nos. 19, 20, 36 and 38. 
     
     
         14 . The method according to  claim 1 , wherein the gene is DR10 type of a HLA-DRB1 gene and the sets of primers are selected from oligonucleotides having nucleotide sequences represented by SEQ ID Nos. 21, 22, 31 and 32. 
     
     
         15 . The method according to  claim 1 , wherein the gene is a HLA-DPA1 gene and the sets of primers are selected from oligonucleotides having nucleotide sequences represented by SEQ ID Nos. 23, 24, 40 and 41. 
     
     
         16 . The method according to  claim 1 , wherein the gene is a HLA-DPB1 gene and the sets of primers are selected from oligonucleotides having nucleotide sequences represented by SEQ ID Nos. 25, 26, 42, 43, 44 and 45. 
     
     
         17 . The method according to  claim 1 , wherein the gene is a HLA-DQA1 gene and the sets of primers are selected from oligonucleotides having nucleotide sequences represented by SEQ ID Nos. 27, 28, 46 and 47. 
     
     
         18 . The method according to  claim 1 , wherein the gene is a HLA-DQB1 gene and the sets of primers are selected from oligonucleotides having nucleotide sequences represented by SEQ ID Nos. 29, 30, 48, 49 and 50. 
     
     
         19 . A primer set for DNA typing of a HLA gene, comprising at least one combination of a forward primer and a reverse primer selected from the groups consisting of the combinations (1) to (17) below:
 (1) a forward primer and a reverse primer selected from oligonucleotides having nucleotide sequences represented by SEQ ID Nos. 1, 2 and 3;   (2) a forward primer and a reverse primer selected from oligonucleotides having nucleotide sequences represented by SEQ ID Nos. 4 and 5;   (3) a forward primer and a reverse primer selected from oligonucleotides having nucleotide sequences represented by SEQ ID Nos. 6, 7 and 8;   (4) a forward primer and a reverse primer selected from oligonucleotides having nucleotide sequences represented by SEQ ID Nos. 9, 10, 11, 31 and 32;   (5) a forward primer and a reverse primer selected from oligonucleotides having nucleotide sequences represented by SEQ ID Nos. 11, 12, 31 and 33;   (6) a forward primer and a reverse primer selected from oligonucleotides having nucleotide sequences represented by SEQ ID Nos. 13, 14, 32 and 34;   (7) a forward primer and a reverse primer selected from oligonucleotides having nucleotide sequences represented by SEQ ID Nos. 15, 16, 31 and 32;   (8) a forward primer and a reverse primer selected from oligonucleotides having nucleotide sequences represented by SEQ ID Nos. 13, 14, 31, 35 and 36;   (9) a forward primer and a reverse primer selected from oligonucleotides having nucleotide sequences represented by SEQ ID Nos. 13, 14, 31, 32 and 37;   (10) a forward primer and a reverse primer selected from oligonucleotides having nucleotide sequences represented by SEQ ID Nos. 17, 18, 36 and 38;   (11) a forward primer and a reverse primer selected from oligonucleotides having nucleotide sequences represented by SEQ ID Nos. 13, 14, 31 and 39;   (12) a forward primer and a reverse primer selected from oligonucleotides having nucleotide sequences represented by SEQ ID Nos. 19, 20, 36 and 38;   (13) a forward primer and a reverse primer selected from oligonucleotides having nucleotide sequences represented by SEQ ID Nos. 21, 22, 31 and 32;   (14) a forward primer and a reverse primer selected from oligonucleotides having nucleotide sequences represented by SEQ ID Nos. 23, 24, 40 and 41;   (15) a forward primer and a reverse primer selected from oligonucleotides having nucleotide sequences represented by SEQ ID Nos. 25, 26, 42, 43, 44 and 45;   (16) a forward primer and a reverse primer selected from oligonucleotides having nucleotide sequences represented by SEQ ID Nos. 27, 28, 46 and 47; and   (17) a forward primer and a reverse primer selected from oligonucleotides having nucleotide sequences represented by SEQ ID Nos. 29, 30, 48, 49 and 50.   
     
     
         20 - 35 . (canceled)