Method for typing of HLA alleles

Abstract

The present invention relates to the typing of HLA alleles. The sequence of exon 2 and exon 3 of the alleles HLA-B*3913, HLA-B*1406, and HLA-B*51new and of exon 2 of the alleles HLA-DRB1*0820, HLA-DRB1*04new and HLA-DRB4*01new are disclosed. The present invention relates to methods for typing of said alleles. According to a preferred embodiment, said typing comprises the following steps: i) amplifying a relevant fragment of said alleles using at least one suitable pair of primers; ii) hybridizing the amplification product of step i) to at least one probe that specifically hybridizes to a target region comprising one or more polymorphic nucleotides in said relevant fragment; iii) determining from the result of step ii) the absence or presence of said alleles in the sample. The present invention further provides primers and probes to be used in said methods for typing. A diagnostic kit comprising said primers and probes is also part of the present invention.

1 . A method for determining the presence or absence of an allele selected from the group consisting of HLA-B*3913, HLA-B*1406 and HLA-B*51new in a sample, wherein exon 2 of said alleles is defined by SEQ ID NO: 22, 72 and 74 respectively, and exon 3 of said allclos is defined by SEQ ID NO: 23, 73 and 75 respectively, wherein said method comprises: 
 a) obtaining nucleic acids from the sample,    b) amplifying nucleic acids comprising part or all of exon 2 and/or part or all of exon 3 of said allele obtained from the sample using at least one suitable pair of primers, and    c) analyzing me amplified nucleic acids for the presence or absence of HLA-B*3913, HLA B*1406 find/or HLA-B*51 5  respectively.    
     
     
         2 . The method according to  claim 1 , wherein the analysis of the amplified nucleic acids comprises: 
 a) hybridizing the amplified nucleic acids to a set of probes which specifically hybridize to target regions within the nucleic adds, which regions comprise one or more polymorphic nucleotides in exon 2 and/or exon 3 of said allele; and    b) determining the absence or presence of the allele HLA-B*3913, HLA-B*1406 and/or HLA-B*51new.    
     
     
         3 . The method according to  claim 2 , wherein said one or more polymorphic nucleotides has a position within exon 2 selected from the group consisting of nucleoude positions: 
 11, 24, 30, 33, 44, 46, 68, 69, 71, 88, 92, 94, 102, 120, 131, 132, 133, 136, 140, 149, 153, 155, 161, 173, 174, 183, 186, 188, 190, 193, 196, 197, 198, 199, 200, 204, 205, 207, 208, 209, 210, 212, 219, 226, 228, 229, 236, 238, 240, 241, 244, 246 and 268, and/or    a position within exon 3 selected from the group consisting of nucleotide positions:    2, 10, 11, 12, 13, 14, 18, 19, 20, 26, 36, 44, 54, 66, 68, 69, 75, 76, 77, 92, 120, 134, 141, 142, 145, 156, 159, 163, 169, 184, 195, 196, 197, 201, 214, 216, 217, 227, 22S, 229 and 240.    
     
     
         4 . The method according to  claim 1 , wherein said primers comprise a nucleotide sequence selected from the group consisting of SEQ ID NO: 26 and 27.  
     
     
         5 . The method according to  claim 2 , wherein said probes comprise a nucleotide sequence selected from the group consisting of: 
 SEQ ID NO: 28-49 for the typing of HLA-B*3913:    SEQ ID NO 29, 30, 32, 37, 41, 47, 48 and 16-89 for the typing of HLA-B*1406; and    SEQ ID NO 33, 44, 79, 84, 87 and 89-102 for the typing of HLA-B*51new.    
     
     
         6 . An isolated nucleic acid comprising exon 2 and/or exon 3 of the allele HLA-B*3913, HLA-B*1406 or HLA-B*51new.  
     
     
         7 . An isolated nucleic acid comprising a sequence selected from the group consisting of: SEQ ID NO: 22, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 23, SEQ ID NO: 73 and SEQ ID NO: 75.  
     
     
         8 . A diagnostic kit for the typing of the alleles HLA-B-i-SQIS, HLA.-B+1406 and/or BLA-B*51 comprising the nucleic acid of  claim 7.