High throughput methods of HLA typing

Abstract

A method for determining an HLA genotype of a subject is disclosed. The method comprises (a) isolating template nucleic acid from the subject; (b) amplifying the template nucleic acid to generate sufficient product for each allele of at least one gene locus to be determined; (c) hybridizing the template nucleic acid with an immobilized array of capture oligonucleotides, each having a known nucleic acid sequence of an HLA allele; and (d) determining the particular capture oligonucleotide to which the template nucleic acid hybridizes, thereby determining the genotype of the subject. A number of additional methods that can eliminate or abbreviate additional steps are also described. Moreover, the present invention provides a method for determining tissue compatibility using the determined HLA genotype.

What is claimed is:  
     
         1 . A method for identifying an HLA genotype of a subject, the method comprising: 
 (a) obtaining a sample comprising a template nucleic acid from said subject;    (b) amplifying said template nucleic acid with a plurality of HLA allele-specific forward primers and HLA allele-specific reverse primers to form amplification products, 
 wherein said forward primers or reverse primers comprise a detectable label;  
   (c) hybridizing said amplification products with a plurality of HLA locus-specific capture oligonucleotides immobilized on a solid phase to form a plurality of detectable complexes; and    (d) detecting said detectable complexes to identify said HLA genotype of said subject.    
     
     
         2 . A method for identifying an HLA genotype of a subject, the method comprising: 
 (a) obtaining a sample comprising a template nucleic acid from said subject;    (b) amplifying said template nucleic acid with a plurality of HLA allele-specific forward primers and HLA allele-specific reverse primers to form amplification products, 
 wherein said forward primers or reverse primers comprise a detectable label;  
   (c) hybridizing said amplification products with a plurality of HLA locus-specific capture oligonucleotides to form a plurality of detectable complexes;    (d) immobilizing said detectable complexes on a solid phase; and    (e) detecting said detectable complexes to identify said HLA genotype of said subject.    
     
     
         3 . The method according to  claim 1  or  2 , wherein said template nucleic acid is isolated from blood or cord blood.  
     
     
         4 . The method according to  claim 1  or  2 , wherein said template nucleic acid is cDNA or genomic DNA.  
     
     
         5 . The method according to  claim 1  or  2 , wherein said solid phase is a member selected from the group consisting of: a bead, a chip, a microtiter plate, a polycarbonate microtiter plate, polystyrene microtiter plate, and a slide.  
     
     
         6 . The method according to  claim 1  or  2 , wherein said HLA genotype is a class I HLA genotype.  
     
     
         7 . The method according to  claim 1  or  2 , wherein said HLA allele-specific forward primers and HLA allele-specific reverse primers are selected from the group consisting of: 
 SEQ ID NOS:1-160.  
 
     
     
         8 . The method according to  claim 1  or  2 , wherein said locus-specific capture oligonucleotides are selected from the group consisting of: 
 SEQ ID NOS:165-168.  
 
     
     
         9 . The method according to  claim 8 , wherein said capture oligonucleotides further comprise a 5′ amine group or a 5′(T) 5-20  oligonucleotide sequence.  
     
     
         10 . The method according to  claim 1  or  2 , wherein said HLA genotype is a class II HLA genotype.  
     
     
         11 . The method according to  claim 1  or  2 , wherein said HLA allele-specific forward primers and HLA allele-specific reverse primers are selected from the group consisting of:selected from the group consisting of: 
 SEQ ID NOS: 169-269.  
 
     
     
         12 . The method according to  claim 1  or  2 , wherein said locus-specific capture oligonucleotides are selected from the group consisting of: 
 SEQ ID NOS: 270-275.  
 
     
     
         13 . The method according to  claim 12 , wherein said capture oligonucleotides further comprise a 5′ amine group or a 5′(T) 5-20  oligonucleotide sequence.  
     
     
         14 . The method according to  claim 1  or  2 , wherein said detectable label comprises a member selected from the group consisting of: 
 radioactive moiety, a fluorescent moiety, a chemiluminescent moiety, an antigen, and a binding protein.  
 
     
     
         15 . The method of  claim 14 , wherein said fluorescent moiety is fluorescein or 5-(2′-aminoethyl) aminonaphtalene-1-sulfonic acid (EDANS).  
     
     
         16 . A method for identifying an HLA genotype of a subject, the method comprising: 
 (a) isolating template nucleic acid from a sample from said subject;    (b) immobilizing a plurality of HLA allele-specific reverse primers on a solid phase;    (c) amplifying said template nucleic acid with a plurality of HLA allele-specific forward primers and said immobilized reverse HLA allele-specific reverse primers to form amplification products, 
 wherein said forward primers comprise a detectable label; and  
   (d) detecting said amplification products to identify said HLA genotype of said subject.    
     
     
         17 . The method according to  claim 16 , wherein said template nucleic acid is cDNA or genomic DNA.  
     
     
         18 . The method according to  claim 16 , wherein said template nucleic acid is isolated from blood or cord blood.  
     
     
         19 . The method according to  claim 16 , wherein said solid phase is a member selected from the group consisting of: a bead, a chip, a microtiter plate, a polycarbonate microtiter plate, polystyrene microtiter plate, and a slide.  
     
     
         20 . The method according to  claim 16 , wherein said HLA genotype is a class I HLA genotype.  
     
     
         21 . The method according to  claim 16 , wherein said HLA allele-specific reverse primers and said HLA allele-specific forward primers are selected from the group consisting of: 
 SEQ ID NOS:1-160.    
     
     
         22 . The method according to  claim 16  wherein said HLA allele-specific reverse primers further comprise a 5′ amine group or a 5′(T) 5-20  oligonucleotide sequence.  
     
     
         23 . The method according to  claim 16 , wherein said HLA genotype is a class II HLA genotype.  
     
     
         24 . The method according to  claim 16 , wherein said HLA allele-specific reverse primers and said HLA allele-specific forward primers are selected from the group consisting of: 
 SEQ ID NOS: 169-269.    
     
     
         25 . The method according to  claim 16 , wherein said detectable label comprises a member selected from the group consisting of: 
 radioactive moiety, a fluorescent moiety, a chemiluminescent moiety, an antigen, and a binding protein.    
     
     
         26 . The method of  claim 25 , wherein said fluorescent moiety is fluorescein or 5-(2′-aminoethyl) aminonaphtalene-1-sulfonic acid (EDANS).  
     
     
         27 . The method of  claim 16 , wherein said forward primers and said reverse primers are selected from the group consisting of: 
 SEQ ID NOS:1-160.    
     
     
         28 . The method of  claim 16 , wherein said forward primers and said reverse primers are selected from the group consisting of: 
 SEQ ID NOS: 169-269.