Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-12-13
2001-11-20
Campbell, Eggerton A. (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200
Reexamination Certificate
active
06319671
ABSTRACT:
FIELD OF THE INVENTION
The present invention is directed to methods for selective and simultaneous hybridization of multiple allele-specific probes to the HLA DQB1 locus. Simultaneous detection of the HLA alleles allows more rapid screening than sequential assay methods. The HLA DQB1 alleles detected by the present invention are associated with susceptibility to, or protection from, insulin dependent diabetes mellitus.
BACKGROUND OF THE INVENTION
The HLA class II gene cluster plays an essential role in immune activation by presenting processed antigens to various lymphoid cell types of the immune system. This role is not only functional during the body's normal defense against invading pathogens, but also during an inappropriate attack on self tissues causing autoimmune diseases. In type I diabetes (IDDM or insulin dependent diabetes mellitus) this autoimmune attack is on the islet cells of the pancreas whose function is to secrete insulin and regulate the body's glucose metabolism. Not all of the hundreds of possible HLA genes are associated with this inappropriate autoimmune attack. Indeed, only two of the 25 known HLA DQB1 genes, namely DQB1 0201 and 0302, are highly associated with the disease in caucasian populations while the DQB1 0303 allele is associated with increased risk for IDDM in the Asian population. In addition, another of these genes, DQB1 0602, has the ability to suppress this greater risk of contracting diabetes when present as part of the heterozygote inheritance with 0302. The rarer DQB1 0202 allele has not been studied with respect to diabetes risk assessment. However, since its DNA sequence is identical to the DQB1 0201 sequence in the region of antigen binding, it is thought to impart the same increased risk as the DQB1 0201 allele. The increased risk of contracting type I diabetes can be as high as 200 fold for people who have inherited both the DQB1 0201 and 0302 genes (see, J. Nepom,
Diabetic Reviews
1:93 (1993)).
U.S. Pat. No. 5,039,606 “Diagnostic Probe for Diabetes Type I Predisposition” filed Oct. 29, 1987 describes a probe sequence which purportedly was specifically reactive with the DQB1 0302 sequences. However, subsequent HLA sequencing (see, Marsh and Bodmer,
Tissue Antigens,
45:258-280 (1995), incorporated by reference herein) has revealed that the probe is not specific to DQB1 0302 allele. In fact, the disclosed probe reacts with nine of the twenty-five known DQB1 alleles, namely 0302, 03032, 0305, 0401, 0402, 05031, 05032, 06011, and 06012.
Accordingly, what is needed in the art are compositions and methods for specifically detecting all of the high risk alleles: DQB1 0201, 0202, 0302, 0303. Additionally, what is needed are compositions and methods for specifically detecting the risk reducing DQB1 allele 0602. More particularly, what is needed are compositions and methods to specifically and simultaneously detect the DQB1 alleles 0201, 0202, 0302, 0303, and 0602 under the uniform low-temperature assay conditions which are desirable for high volume clinical testing. Quite surprisingly, the present invention provides these and other advantages.
SUMMARY OF THE INVENTION
In one aspect, the present invention relates to a method of detecting the presence of HLA DQB1 alleles 0201/0202, 0302/0303, and 0602 in a human biological sample. The methods comprises the steps of contacting, under stringent conditions, an amplicon comprising a sequence from codon 23 through 40 of the human leukocyte antigen (HLA) DQB1 gene with at least three unique human leukocyte antigen (HLA) DQB1 allele-specific probes to form a hybridization complex and detecting the hybridization complexes as an indication of the presence of the HLA DQB1 alleles 0201/0202, 0302/0303, and 0602. In this aspect of the invention the amplicon is no more than 2000 base pairs in length and is amplified with at least two primers, wherein one of the two primers selectively hybridizes, under stringent conditions, to the same nucleic acid sequence of the HLA DQB1 allele as a primer having the sequence 5′-GGAGCGCGTGCGTCTTGTG-3′ (SEQ ID NO:7). Further, the HLA DQB1 allele-specific probe is between 15 and 200 nucleotides in length, and each of the DQB1 allele-specific probes includes a nucleic acid segment of at least 15 contiguous nucleotides which selectively hybridizes under stringent conditions to the same nucleic acid sequence of the HLA DQB1 allele as an oligonucleotide selected from the group consisting of SEQ ID NOS: 2, 4, 6, and complementary sequences thereof. Each of the allele-specific probes selectively hybridizes under the same stringent hybridization conditions.
In some embodiments the amplicon is no longer than 1000 base pairs in length, in other embodiments the amplicon is no longer than 700 base pairs in length. In further embodiments, the nucleic acid segments selectively hybridize under stringent conditions to at least 18 contiguous bases of an oligonucleotide selected from the group consisting of: SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, and complementary sequences thereof. In preferred embodiments, the nucleic acid segment is complementary to an oligonucleotide selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, and complementary sequences thereof. In a particularly preferred embodiment, the amplicon further comprises a sequence having any number of contiguous bases in the sequence from codon 22 to 41. Preferably, the amplicon is generated using a primer which selectively hybridizes, under stringent conditions, to the same nucleic acid sequence of the HLA DQB1 allele as a primer having the sequence 5′-TCTGGCTGTTCCAGTACTCGGC-3′ (SEQ ID NO:8) in conjunction with a primer which selectively hybridizes, under stringent conditions, to the same nucleic acid sequence of the HLA DQB1 allele as a primer having the sequence shown in SEQ ID NO:7.
In another aspect, the present invention relates to a method of detecting the presence of HLA DQB1 alleles 0201/0202, 0302/0303, and 0602 in a human biological sample. The methods comprises the steps of contacting an amplicon comprising a sequence from codon 23 through 49 of the human leukocyte antigen (HLA) DQB1 gene with at least three unique human leukocyte antigen (HLA) DQB1 allele-specific probes to form a hybridization complex and detecting the hybridization complexes as an indication of the presence of the HLA DQB1 alleles 0201/0202, 0302/0303, and 0602. In this aspect of the invention, the amplicon is no more than 2000 base pairs in length and is amplified with at least two primers, wherein one of the two primers selectively hybridizes, under stringent conditions, to the same nucleic acid sequence of the HLA DQB1 allele as a primer having the sequence 5′-GGAGCGCGTGCGTCTTGTG-3′ (SEQ ID NO:7). Further, the HLA DQB1 allele-specific probes is between 15 and 200 nucleotides in length, and each of the DQB1 allele-specific probes includes a nucleic acid segment of at least 15 contiguous nucleotides which selectively hybridizes under stringent conditions to the same nucleic acid sequence of the HLA DQB1 allele as an oligonucleotide selected from the group consisting of SEQ ID NOS: 1, 2, 3, 4, 5, 6, and complementary sequences thereof, with the proviso that each of the allele-specific probes selectively hybridizes under the same stringent hybridization conditions.
In some embodiments the amplicon is no longer than 1000 base pairs in length, in other embodiments the amplicon is no longer than 700 base pairs in length. In other embodiments, the nucleic acid segments selectively hybridize under stringent conditions to at least 18 contiguous bases of an oligonucleotide selected from the group consisting of: SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, and complementary sequences thereof. In preferred embodiments, the nucleic acid segment is complementary to an oligonucleotide selected from the group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, and complementary sequences thereof. In a particularly preferred embodiment, the amplicon furth
Gersuk Vivian H.
Howard Sharon
U'ren Jack
Campbell Eggerton A.
Saigene Corporation
Townsend and Townsend / and Crew LLP
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