Method for typing of minor histocompatibility antigen HA-1

Abstract

The present invention provides a method for typing of alleles of the Minor Histocompatibility Antigen HA-1 in a sample, the method comprising: a) contacting the genomic polynucleic acids in the sample with at least one pair of primers, whereby the 5′- and/or the 3′-primer of the at least one pair of primers specifically hybridize to target regions comprising polymorphic nucleotides in said alleles, and performing an amplification reaction; b) for each of the at least one pair of primers detecting whether or not in step a) an amplification product is formed; c) inferring from the result of step b) which HA-1 allele is present in the sample. The present invention also provides a method for genomic typing of alleles of the Minor Histocompatibility Antigen HA-1 in a sample and primers and probes for use in the above-mentioned methods. Diagnostic kits enabling the methods are also provided.

1 . A method for typing of alleles of the Minor Histocompatibility Antigen HA-1 in a sample, the method comprising detecting polymorphic nucleotides in cDNA or genomic nucleic acids of the alleles contained in the sample.  
     
     
         2 . A method according to  claim 1 , wherein the alleles of the Minor Histocompatibility Antigen HA-1 are the H allele and the R allele as shown in  FIG. 5 .  
     
     
         3 . A method for genomic typing according to  claim 1 , the method comprising: 
 a) contacting the genomic polynucleic acids in the sample with at least one pair of primers, whereby the 5′- and/or the 3′-primer of the at least one pair of primers specifically hybridize to target regions comprising polymorphic nucleotides in the alleles, and performing an amplification reaction;    b) for each of the at least one pair of primers, detecting whether or not in step a) an amplification product is formed;    c.) inferring from the result of step b) which HA-1 allele is present in the sample.    
     
     
         4 . A method according to  claim 1 , wherein 
 the at least one pair of primers comprises a 5′-primer that specifically hybridizes to a target region comprising the nucleotides at position 4 or at positions 4 and 8 in the HA-1 allele; or, wherein    the at least one pair of primers comprises a 3′-primer that specifically hybridizes to a target region comprising the nucleotides at position 8 or at positions 4 and 8 in the HA-1 allele, with the positions as shown in  FIG. 5 .    
     
     
         5 . A method according to  claim 4 , wherein 
 the 5′-primer is combined with a 3′-primer specifically hybridizing to a target region in intron a; and/or wherein    the 3′-primer is combined with a 5′-primerspecifically hybridizing to a target region in exon a, with intron a and exon a as shown in  FIG. 5 .    
     
     
         6 . A method according to any of claims  1 , wherein the primer has SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, or SEQ ID NO 7.  
     
     
         7 . A method for genomic typing according to  claim 1 , the method comprising: 
 a) amplifying a fragment of the alleles, with the fragment comprising at least one polymorphic nucleotide, by use of at least one pair of primers specifically hybridizing to conserved target regions in the alleles;    b) hybridizing the amplified product of step a) to at least one probe specifically hybridizing to a target region comprising one or more polymorphic nucleotides in the alleles;    c) inferring from the result of step b) which HA-1 allele is present in the sample.    
     
     
         8 . A method according to  claim 7 , wherein the at least one pair of primers comprises a 5′-primer specifically hybridizing to a conserved target region in exon a and/or a 3′-primer specifically hybridizing to a conserved target region in intron a, with exon a and intron a being indicated in  FIG. 5 .  
     
     
         9 . A method according to  claim 7 , wherein the at least one probe specifically hybridizes to a target region comprising the nucleotides at position 4 and/or 8 in the HA-1 allele, with the positions being indicated in  FIG. 5 .  
     
     
         10 . A method according to  claim 7 , wherein the primer is SEQ ID NO 2, SEQ ID NO 8, SEQ ID NO 9, or SEQ ID NO 10, and/or the probe is SEQ ID NO 1, SEQ ID NO 12, SEQ ID NO 13, SEQ ID NO 14, SEQ ID NO 15, or SEQ ID NO 16.  
     
     
         11 . A primer for use in genomic typing of alleles of the Minor Histocompatibility Antigen HA-1 according to  claim 7 , wherein the primer includes SEQ ID NO 2, SEQ ID NO 8, SEQ ID NO 9, or SEQ ID NO 10.  
     
     
         12 . A probe for use in genomic typing of alleles of the Minor Histocompatibility Antigen HA-1 in accordance with  claim 7 , wherein the probe includes SEQ ID NO 1, SEQ ID NO 12, SEQ ID NO 13, SEQ ID NO 14, or SEQ ID NO 15.  
     
     
         13 . An isolated polynucleic acid for use in genomic typing of alleles of the Minor Histocompatibility Antigen HA-1 in accordance with  claim 7 , as identified by SEQ ID NO 1, or SEQ ID NO 17, or SEQ ID NO 18 or an isolated polynucleic acid displaying at least 80% sequence homology to one of the foregoing polynucleic acids, or a fragment of one of the foregoing polynucleic acids.  
     
     
         14 . A method for genomic typing of alleles of the Minor Histocompaubility Antigen HA-1 according to  claim 1  com