# US 5,702,885 A · Method for HLA typing

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- Number: US 5,702,885 A
- Document ID: US5702885A
- Status: Granted
- Kind: A
- Application number: 08/057,957
- Filed: 1993-04-08
- Granted: 1997-12-30
- Earliest claimed: 1990-06-27
- Assignees: Blood Center Research Foundation
- Inventors: Lee Ann Baxter-Lowe; Jack A. Gorski
- Web page: https://patentgrep.com/patent/US5702885A

## CPC

- C12Q 1/6881: for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
- C07K 14/70539: {MHC-molecules, e.g. HLA-molecules}
- C12Q 1/6813: Hybridisation assays
- C12Q 2600/156: Polymorphic or mutational markers
- C12Q 2600/172: Haplotypes
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)

## Abstract

A method for HLA typing by amplification of a sample followed by sequence-specific oligonucleotide hybridization with a labelled oligonucleotide probe provides for both positive and negative controls. Control sequences representing known allelic polymorphisms at the locus in question are subjected to the labelled probe along with the sample. This method reduces errors and improves the chance of obtaining a successful tissue match, as is vital in the case of tissue transplants, particularly bone marrow transplants. Probes and PCR primers useful in HLA-DR typing are also provided.

## Claims

### Claim 1

An oligonucleotide probe consisting of:
an oligonucleotide sequence which undergoes sequence-specific hybridization with an HLA-DR beta consensus sequence at positions 61-64 characterized by the sequence consisting of 5'-GGAACAGCCAGA, and
a labelling substance other than a nucleotide sequence, which labelling substance facilitates detection of the probe.

### Claim 2

An oligonucleotide primer useful in conducting a polymerase chain reaction on HLA-DR beta DNA, consisting of the sequence 5'-ATTTCTTCAATGGGACGGAGC.

### Claim 3

An oligonucleotide primer useful in conducting a polymerase chain reaction on HLA-DR beta DNA, consisting of the sequence 5'-CGCCGCTGCACTGTGAAGCTCTC.

### Claim 4

An oligonucleotide primer useful in conducting a polymerase chain reaction on HLA-DR beta DNA, consisting of the sequence 5'-GTCCCCACAGCACGTTTCTTG.

### Claim 5

In a method for HLA typing including the steps of amplifying an HLA sequence of DNA of a subject, bringing a first oligonucleotide probe that recognizes an allelic polymorphism at a selected HLA locus into contact with a first sample of the amplified HLA sequence, and detecting the formation of DNA duplexes by the first probe and the amplified HLA sequence in order to determine that the allelic polymorphism recognized by the first probe is present in the amplified HLA sequence, the improvement which comprises, at the same time and under the same conditions as used for bringing the first oligonucleotide probe into contact with the first sample of the amplified HLA sequence:
separately bringing a second oligonucleotide probe into contact with a second sample of the amplified HLA sequence, which second probe undergoes specific hybridization with an HLA sequence known to be found in the amplified HLA sequence and which is different from the sequence that the first probe hybridizes with; and
detecting a false negative by observation of a failure to hybridize between the first probe and the amplified HLA sequence together with a failure to hybridize between the second probe and the amplified HLA sequence;
wherein the first probe recognizes an HLA-DR beta allelic polymorphism and the second probe consists of the oligonucleotide sequence 5'-GGAACAGCCAGA or the complement thereof, and a labelling substance other than a nucleotide sequence, which labelling substance facilitates detection of the probe.

### Claim 6

In a method for HLA typing including the steps of amplifying an HLA sequence of DNA of a subject, bringing a first oligonucleotide probe that recognizes an allelic polymorphism at a selected HLA locus into contact with a first sample of the amplified HLA sequence, and detecting the formation of DNA duplexes by the first probe and the amplified HLA sequence in order to determine that the allelic polymorphism recognized by the first probe is present in the amplified HLA sequence, the improvement which comprises, at the same time and under the same conditions as used for bringing the first oligonucleotide probe into contact with the first sample of the amplified HLA sequence:
separately bringing a second oligonucleotide probe into contact with a second sample of the amplified HLA sequence, which second probe undergoes specific hybridization with an HLA sequence known to be found in the amplified HLA sequence and which is different from the sequence that the first probe hybridizes with; and
detecting a false negative by observation of a failure to hybridize between the first probe and the amplified HLA sequence together with a failure to hybridize between the second probe and the amplified HLA sequence;
wherein the first probe recognizes an HLA-DO beta allelic polymorphism and the second probe consists of the oligonucleotide sequence 5'-CTATAACCGAGA or the complement thereof, and a labelling substance other than a nucleotide sequence, which labelling substance facilitates detection of the probe.

### Claim 7

In a method for HLA typing including the steps of amplifying an HLA sequence of DNA of a subject, bringing a first oligonucleotide probe that recognizes an allelic polymorphism at a selected HLA locus into contact with a first sample of the amplified HLA sequence, and detecting the formation of DNA duplexes by the first probe and the amplified HLA sequence in order to determine that the allelic polymorphism recognized by the first probe is present in the amplified HLA sequence, the improvement which comprises, at the same time and under the same conditions as used for bringing the first oligonucleotide probe into contact with the first sample of the amplified HLA sequence:
separately bringing a second oligonucleotide probe into contact with a second sample of the amplified HLA sequence, which second probe undergoes specific hybridization with an HLA sequence known to be found in the amplified HLA sequence and which is different from the sequence that the first probe hybridizes with; and
detecting a false negative by observation of a failure to hybridize between the first probe and the amplified HLA sequence together with a failure to hybridize between the second probe and the amplified HLA sequence;
wherein the first probe recognizes an HLA-B allelic polymorphism and the second probe consists of the oligonucleotide sequence 5'-GGAGGGGCCGGAGTATTGG or the complement thereof, and a labelling substance other than a nucleotide sequence, which labelling substance facilitates detection of the probe.

### Claim 8 (depends on 5)

The method of claim 5, wherein the first and second probes are deposited on a reaction surface having the amplified HLA sequence disposed thereon.