Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1996-05-21
2003-01-07
Priebe, Scott D. (Department: 1632)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200
Reexamination Certificate
active
06503707
ABSTRACT:
This invention was made with Government support, and the Government has certain rights in the invention.
FIELD OF THE INVENTION
The invention relates to a method for genetic typing, particular to a method for identifying nucleotide sequences of polymorphic genes such as genes coding for human leukocyte antigens.
BACKGROUND OF THE INVENTION
The major histocompatibility complex of humans is a cluster of genes occupying a region located on the sixth chromosome. Human leukocyte antigen (HLA) genes are highly polymorphic and code for human leukocyte antigens whose structural variation is a major factor influencing tissue transplantation, immunity and autoimmunity. The polymorphic HLA proteins have been designated HLA-A, -B, -C, -DR, -DQ and -DP. The HLA-A, -B, and -C proteins are described as class I HLA proteins and are characterized by a polymorphic chain, alpha, and a nonpolymorphic chain, beta 2 microglobulin. The HLA-DR, -DQ and -DP proteins are classified as class II HLA proteins and are also comprised of two polypeptide chains, an alpha chain and a highly polymorphic beta chain. These HLA-D-region proteins are encoded by loci designated HLA-DRA, -DRB1, -DRB3, -DRB4, -DRB5, -DQA1, -DQB1, -DPA1 and -DPB1. HLA-DRA, -DQA1 and -DPA1 are much less polymorphic than HLA-DRB1, DQB1 and -DPB1.
The HLA proteins encoded by the polymorphic genes of the different HLA loci have previously been typed by serological methods. The major drawbacks to such HLA typing, are the complexity of the sera and the lack of widespread availability of standard sera necessary to conduct the tests. Serological HLA-typing techniques require the presence of detectable levels of HLA proteins on the surface of lymphocytes. In some cases, such as HLA-deficient severe combined immunodeficiency (SCID) and cellular depletion due to chemotherapy, the levels of the HLA proteins are inadequate to achieve reliable HLA typing. Another limitation of traditional serological typing methods is the inability to resolve all functionally important HLA proteins. These circumstances have prompted the development of methods for analysis of HLA polymorphism at the genetic level, as described by Bidwell,
Immunology Today
9: 18-23 (1988), and by Angelini et al., Proc.
Nat'l Acad. Sci., USA
83: 4489-93 (1986).
Non-serological HLA typing methods have been proposed to overcome drawbacks with serological typing. One such method involves the use of DNA restriction fragment length polymorphism (RFLP) as a basis for HLA typing. See Erlich, U.S. Pat. No. 4,582,788, Opelz et al.,
Lancet
338:461-463 (1991) and Mickelson et al. Tissue Antigens 41:86-93 (1993). RFLP analysis, however, fails to differentiate between certain alleles that are known to exist in the population (e.g., subtypes of HLA-DR4), and thus, cannot be used to distinguish certain combinations of alleles. Moreover, its practical usefulness is limited because the procedures involved are laborious, and difficulties arise in interpreting data for certain combinations of alleles.
Some typing methods, including RFLP-based analyses, utilize labelled oligonucleotides to identify specific HLA nucleotide sequences. In particular, the use of oligonucleotide probes has been found advantageous in HLA-DR typing to identify HLA-proteins which are not detectable serologically. See Angelini et al., supra; Scharf et al.,
Science
233: 1076-78; Cox et al.,
Am. J. Hum. Gen
., 43: 954-63 (1988); Tiercy et al., Proc.
Nat'l Acad. Sci. USA
85: 198-202 (1988),
Human Immunol
. 24: 1-14 (1989). For example, sequence-specific oligonucleotide probe hybridization (SSOPH) can discriminate single base pair mismatches, which is equivalent to detecting a single amino acid polymorphism in HLA proteins.
More recently, utilizing the polymerase chain reaction (PCR) process, as described in U.S. Pat. No. 4,683,202, researchers have used sequence-specific oligonucleotide (“SSO”) probe hybridization to perform HLA-Class II typing. The method entails amplifying a polymorphic region of an HLA locus using PCR, contacting the amplified DNA to a sequence-specific oligonucleotide probes under hybridizing conditions, and detecting hybrids formed between the amplified DNA and the sequence-specific oligonucleotide probes. Alleles of all class II HLA and some class I HLA genes have been identified in the aforementioned manner. See Saiki et al.,
Nature
, 324:163-166, 1986, Bugawan et al.,
J. Immunol
., 141:4024-4030, 1988, and Gyllensten et al.,
Proc. Natl. Acad. Sci. USA
, 85:7652-7656, 1988.
In the parent application, Ser. No. 08/025,038, filed Mar. 1, 1993, a two-step method is described for resolving HLA-DQB1 and HLA-DRB1 alleles more fully. The described two-step method comprises a low resolution, locus-specific amplification assay followed by a high resolution, intra-locus specific assay. Although highly accurate, the two-step method is consuming and labor intensive because it requires at least two amplification steps. See also Molkentin et al.,
Hum. Immunol
., 31:114, 1991. A need exists, therefore, for a more rapid and easily implemented approach to identifying nucleotide sequences of polymorphic genes, and in particular, for identifying nucleotide sequences of polymorphic genes coding for human leukocyte antigens. Solid supports such as Dynabeads (microbeads available from Dynal) have been used for purification of mixtures prior to sequencing. See Hulman et al.
Nucl. Acids. Res
. 17:4937-4946 (1989) and Fry et al.,
BioTechniques
Vol. 13, No. 1, p. 124-131 (1992). However, no such method has been used for the purpose of typing an unknown sample.
SUMMARY OF THE INVENTION
A method for genetic typing according to the invention includes the steps amplifying a genetic sequence, which genetic sequence may occurs naturally in two or more genetic types, separating from a mixture of amplified DNA a sample of one type substantially free of DNA of the other types, and then optionally further analyzing the sample to confirm or further characterize the DNA of the selected type. The genetic type will commonly be a basic type that includes two or more specific alleles, in which case the step of further analyzing the sample may further entail determining which allele of the basic type is present. HLA subtypes that can be differentiated using a single oligonucleotide probe, such as HLA-DR1, DR2, DR3, etc. represent a basic genetic type within the meaning of the invention. For purposes of typing according to the invention, references to DNA should also be understood to include RNA sequences, for which it is generally necessary to first prepare the corresponding cDNA.
A preferred method for genetic typing according to the invention includes the steps of:
(a) amplifying a genetic sequence of a subject to obtain amplified DNA, which genetic sequence occurs naturally in two or more genetic types;
(b) bringing a sample of the amplified DNA into contact with an oligonucleotide probe bound to a support under stringent hybridizing conditions, which oligonucleotide probe hybridizes specifically with DNA having a sequence of one of the genetic types and not with DNA having a sequence of the other genetic types;
(c) removing unbound amplified DNA, for example, by washing the support; and
(d) analyzing the sample to determine if the one genetic type is present.
“Genetic type” as used herein refers to virtually any naturally occurring nucleotide sequence which exists in a number of variations or alleles. HLA types, such as HLA DP, DQ and DR alleles, are examples. Amplification as carried out in step (a) need be only locus specific, not sequence specific. Step (a) may also be carried out using the combined products of two or more locus-specific amplifications, or the combined products of two or more sequence-specific amplifications. For purposes of the present invention, locus-specific amplification refers to amplification of all or a major portion of a locus such that most if not all naturally-occurring alleles within that locus would be amplified. Primers for locus-specific amplification have sequences corresponding to si
Meyers Philip G.
Priebe Scott D.
The Blood Center Research Foundation, Inc.
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