High throughput methods of HLA typing

• US 6,670,124 B1
• Filed: 12/20/2000
• Issued: 12/30/2003
• Est. Priority Date: 12/20/1999
• Status: Expired due to Fees

• Current Assignee: Stemcyte Inc
• Original Assignee: Stemcyte Inc
• Inventors: Chow, Robert, Tonai, Richard
• Primary Examiner(s): Fredman, Jeffrey
• Assistant Examiner(s): SWITZER, JULIET CAROLINE
• Application Number:
  US09/747,391
• Time in Patent Office: 1,105 Days
• Field of Search: 435/6, 435/91.2, 536/24.3, 536/23.1, 536/24.31
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/6881 for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers

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.

Claims

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 an HLA locus-specific capture oligonucleotide immobilized on a solid phase to form a plurality of detectable complexes, wherein the HLA locus-specific capture oligonucleotide comprises SEQ ID NO;

277; and

(d) detecting said detectable complexes to identify said HLA genotype of said subject.

2. The method according to claim 1, wherein said capture oligonucleotides further comprise a 5′

amine group or a 5′

(T)5-20 oligonucleotide sequence.

3. The method of claim 1, wherein the HLA locus-specific capture oligonucleotide consists of SEQ ID NO:

277.

4. The method according to claim 3, wherein said capture oligonucleotides further comprise a 5′

amine group or a 5′

(T)5-20 oligonucleotide sequence.

5. 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 an HLA locus-specific capture oligonucleotide to form a plurality of detectable complexes, wherein the HLA locus-specific capture oligonucleotide comprises SEQ ID NO;

277;

(d) immobilizing said detectable complexes on a solid phase; and

(e) detecting said detectable complexes to identify said HLA genotype of said subject.

6. The method according to claim 5, wherein said capture oligonucleotides further comprise a 5′

amine group or a 5′

(T)5-20 oligonucleotide sequence.

7. The method of claim 5, wherein the HLA locus-specific capture oligonucleotide consists of SEQ ID NO:

277.

8. The method according to claim 7, wherein said capture oligonucleotides further comprise a 5′

amine group or a 5′

(T)5-20 oligonucleotide sequence.