Beta 2 adrenergic polymorphism detection

Details Beta 2 adrenergic polymorphism detection Beta 2 adrenergic polymorphism detection

2001-10-09

2003-07-15

Myers, Carla J. (Department: 1634)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S091200, C435S810000, C536S023500, C536S024310, C536S024330

Reexamination Certificate

active

06593092

ABSTRACT:

TECHNICAL FIELD
The present invention relates to nucleic acid polymorphisms and, in particular, relates to detecting a single nucleotide polymorphism using nucleic acid amplification technology.
BACKGROUND OF THE INVENTION
Studies designed to determine the sequence of the human genome, as well as studies designed to compare human genomic sequences, have elicited information regarding polymorphisms in the human genome. A wide variety of polymorphisms in the human genome have previously been described. The various types of human genetic polymorphisms include single base substitutions; insertions or deletions; variable numbers of tandem repeats; deletions of all or a large part of a gene; gene amplifications; and chromosomal rearrangements. Generally, polymorphisms that involve a single nucleotide are called single nucleotide polymorphisms (“SNPs”).
Recently, a SNP in codon 16 of the &bgr;2 adrenergic receptor gene has been reported and associated with a variation in response to &bgr; agonist therapy (Drazen, J M et al, Thorzx, 1996, 51:1168; Liggett, S. B., Am. J. Respir. Crit. Care Med., 1997, 156:S156-62; Martinez, F. D. et al, J. Clin. Invest., 1997, 100:3184-8). Adrenergic receptors are hormone receptors on the surfaces of various cells. When bound to an adrenergic receptor site, a hormone can trigger a cascade of cellular events. Hence, adrenergic receptors and the hormones that bind to them, in large part form the mechanism that controls cellular events at a molecular level. Many pharmacological compounds mimic molecules that bind to adrenergic receptor sites and, in this manner, clinically regulate cellular function. For example, a class of drugs known as beta-agonists bind to &bgr;2 adrenergic receptor sites and are widely used as a medication for asthma. Individuals with a SNP in codon 16 of the &bgr;2 adrenergic gene, however, may not respond to such therapies due to a conformational, or other, change in the receptor that causes a decrease in the affinity between the receptor and the medication or hormone.
It would be advantageous, therefore, to provide a means for detecting the polymorphism in codon 16 of the &bgr;2 adrenergic receptor gene prior to prescribing medications that would not be efficacious as a result of the polymorphism.
SUMMARY OF THE INVENTION
Provided herein are methods capable of analyzing polymorphic nucleic acid sequences in a manner suitable for automation. The present invention provides reagents, methods, and kits for amplifying and detecting a target sequence having a polymorphism at codon 16 of the &bgr;2 adrenergic receptor gene in a test sample. In particular, SEQ ID NO: 2 and SEQ ID NO: 3 can be employed as amplification primers to amplify the target sequence designated herein as SEQ ID NO: 1. It was discovered that these primers specifically and sensitively produce an amplification product that is amenable to detection with SEQ ID NOs: 4, 5, and with molecular beacon probes comprising SEQ ID NOs: 6, 7, 8, and 9. SEQ ID NO: 4 is an internal hybridization probe specific for the wild-type sequence and SEQ ID NO: 5 is an internal hybridization probe specific for the variant (polymorphic) sequence. Similarly, SEQ ID NOs: 6 and 7 are the nucleotide sequence incorporable into a molecular beacon probe for the wild-type sequence, while SEQ ID NOs: 8 and 9 are nucleotide sequences incorporable into molecular beacon probes selective for the variant (polymorphic) sequences.
The target sequence, designated herein as SEQ ID NO: 1, can be amplified by forming a reaction mixture comprising nucleic acid amplification reagents, a test sample containing a target sequence, and primers designated SEQ ID NOs. 2 and 3. Following amplification, the amplified target sequence can be detected. For example, the probes designated SEQ ID NOs: 4 and 5, or the molecular beacon probes incorporating the sequences designated SEQ ID NOs: 6, 7, 8 and 9, can be employed to hybridize to the amplified target sequence to form a probe/amplification product hybrid, which can be detected using any suitable technique selected from a variety of well known techniques. Hence, detecting a probe/amplification product hybrid wherein the probe is SEQ ID NO: 4 indicates the presence of the wild-type sequence. On the other hand, detecting of a probe/amplification product hybrid wherein the probe is SEQ ID NO: 5 would indicate the presence of the polymorphic sequence. Similarly, detecting a signal indicative of the target-bound state from the molecular beacon probe comprising the nucleotide sequence designated SEQ ID NO: 6 or SEQ ID NO: 7 in the presence of the amplification product indicates the presence of the wild-type sequence, whereas detecting a signal indicative of the target-bound state from one of the molecular beacon probes comprising the nucleotide sequence designated SEQ ID NO: 8 or SEQ ID NO: 9 in the presence of the amplification product indicates the presence of the variant (polymorphic) sequence.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides reagents, methods, and kits for amplifying and detecting a target sequence in a test sample. In particular, SEQ ID NO: 2 and SEQ ID NO: 3 can be employed as amplification primers to amplify a nucleic acid sequence potentially comprising the polymorphism in codon 16 of the &bgr;2 adrenergic receptor gene. Hence, both the wild-type and polymorphic version of the target sequence can be amplified using SEQ ID NOs:2 and 3. The sequence AACGGCAGCG CCTTCTTGCT GGCACCCAAT AGAAGCCATG CGCCGGACCA CGACGTCACG CAGCAAAGGG ACGAGGTGTG GGTGGTGGGC ATGGGCATCG TCATGT (SEQ ID NO: 1) is presented as a representative target sequence. Probe sequences, having SEQ ID NO: 4 or SEQ ID NO: 5 can be employed to detect or distinguish the amplification product produced by primers designated SEQ ID NO: 2 and SEQ ID NO: 3 (e.g., indicate the presence of the wild-type or polymorphic sequence in the test sample). Similarly, molecular beacon probes incorporating the nucleotide sequences designated SEQ ID NOs: 6, 7, 8, and 9 can be employed to detect and/or distinguish the amplification product produced by primers designated SEQ ID NOs: 2 and 3 (e.g., indicate the presence of the wild-type or polymorphic sequence in the test sample).
Nucleotide sequences useful in the context of the present invention include:
SEQ ID NO: 2: aacggcagcg ccttcttgc

SEQ ID NO: 3: acatgacgat gcccatgcc

SEQ ID NO: 4: caatagaagc catgc

SEQ ID NO: 5: cccaatggaa gcc

SEQ ID NO: 6: cgtccgcacc caatagaagc catcggacg,

SEQ ID NO: 7: cgtccgatgg cttctattgg gtgcggacg,

SEQ ID NO: 8: cgtccgcacc caatggaagc catcggacg, and

SEQ ID NO: 9: cgtccgatgg cttccattgg gtgcggacg,
as well as artificial analog sequences wherein one or more of the naturally-occurring nucleotides identified above is replaced with a synthetic analog of the nucleotide. In three preferred embodiments, the molecular beacons comprising the nucleotide sequences designated SEQ ID NOs: 6, 7, 8, and 9 consist entirely or essentially of these nucleotide sequences, linked to fluorescein and dabcyl. Molecular beacon probes comprising the nucleotides sequences designated SEQ ID NO: 6 and SEQ ID NO: 9 are preferred to molecular beacon probes comprising SEQ ID NO: 7 and SEQ ID NO: 8, respectively.
The primer, probe, and nucleotide sequences of the molecular beacons disclosed herein, can comprise deoxyribonucleic acid (DNA), ribonucleic acid (RNA) or nucleic acid analogs, such as uncharged nucleic acid analogs including but not limited to peptide nucleic acids (PNAs) which are disclosed in International Patent Application WO 92/20702 or morpholino analogs which are described in U.S. Pat. Nos. 5,185,444, 5,034,506, and 5,142,047 all of which are herein incorporated by reference, as well as other nucleic acid analogs known in the art. For example, the skilled artisan will recognize that where the oligonucleotide designated as T (i.e., thymidine) is indicated, this oligonucleotide also designates U in embodiments where RNA is employed rather than DNA, and designates a nucleic acid analog where non-naturally occurring nucleotide residues

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