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  Methods and compositions for nucleic acid analysisUSPTO Application #: 20070122839Title: Methods and compositions for nucleic acid analysis Abstract: The present invention relates to improved methods and composition for nucleic acid analysis. In particular, the present invention provides improved methods and compositions for carrying out nucleic acid analysis using modified nucleotides. (end of abstract)
Agent: Medlen & Carroll, LLP - San Francisco, CA, US Inventors: Mary Ann Brow, David A. Casimir USPTO Applicaton #: 20070122839 - Class: 435006000 (USPTO) Related Patent Categories: Chemistry: Molecular Biology And Microbiology, Measuring Or Testing Process Involving Enzymes Or Micro-organisms; Composition Or Test Strip Therefore; Processes Of Forming Such Composition Or Test Strip, Involving Nucleic Acid The Patent Description & Claims data below is from USPTO Patent Application 20070122839. Brief Patent Description - Full Patent Description - Patent Application Claims
[0001] The present Application is a Divisional application of co-pending application Ser. No. 10/969,032, filed Oct. 20, 2004, which claims priority to Provisional Application Ser. No. 60/512,638, filed Oct. 20, 2003, each of which is incorporated herein by reference in its entirety. FIELD OF THE INVENTION [0002] The present invention relates to improved methods and composition for nucleic acid analysis. In particular, the present invention provides improved methods and compositions for carrying out nucleic acid analysis using modified nucleotides. BACKGROUND OF THE INVENTION [0003] The development and use of modified nucleotides has expanded the range of nucleic acid analysis techniques. For example, the modified non-natural nucleotides of Eragen Corporation expand the alphabet of bases that may be used in nucleic acid technologies (See e.g., U.S. Pat. Nos. 5,216,141, 5,432,272, 5,958,702, 5,965,364, 6,001,983, 6,037,120, and 6,140,496 and patent application Ser. Nos. 09/415,966, 09/538,338, 09/993,757, 60/205,712, 60/240,398, 60/282,831, 60/240,397, 60/252,783, and 60/253,382, each of which is incorporated herein in its entirety). While these modified nucleotides have been found useful, compositions and methods that provide improved sensitivity and flexibility in using such products in nucleic acid analysis methods are needed. [0004] Modified nucleotides also find use in nucleic acid amplification reactions such as the polymerase chain reaction. See, e.g., U.S. Pat. No. 09/861,292, published as Publication No. 2002/0150900, and U.S. Pat. No. 10/977,615, published as Publication No. 2004/0106108, each of which is incorporated herein by reference in its entirety. While these applications have been found useful, methods and compositions that reduce background signal and improve assay sensitivity are needed. SUMMARY OF THE INVENTION [0005] The present invention relates to improved methods and compositions for nucleic acid analysis. In particular, the present invention provides improved methods and compositions for carrying out nucleic acid analysis using modified nucleotides. Still more particularly, the present invention provides improved methods and compositions for carrying out nucleic acid analysis using non-naturally occurring nucleotides that are complementary to other non-naturally occurring nucleotides but that are capable of base-pairing to one or more naturally occurring nucleotides. [0006] In some embodiments, the present invention provides a kit comprising an extension-disabled non-naturally occurring nucleotide. In some preferred embodiments, the kit further comprises one or more of an oligonucleotide primer, a polymerase (e.g., a DNA polymerase, a thermostable polymerase, a polymerase lacking 5' nuclease activity, etc.), and reagents for conducting a polymerase chain reaction. In some preferred embodiments, the extension-disabled non-naturally occurring nucleotide comprises a dideoxy nucleotide (e.g., a dideoxy iso-G). [0007] The present invention further provides methods for analyzing a target nucleic acid, comprising: exposing a target nucleic acid to polymerase, a first amplification primer having a first non-natural nucleotide, a second amplification primer, and an extension-disabled second non-natural nucleotide complementary to said first non-natural nucleotide under conditions such that an extension product is generated from at least said second amplification primer, wherein said extension product incorporates said second non-natural nucleotides. In some embodiments, the first and second amplification primers are selected to hybridize to a portion of said target nucleic acid to define an amplification region, wherein said amplification region is selected to minimize the presence of T nucleotides in said amplification region. [0008] In some embodiments, the present invention provides improved methods of performing primer-directed amplification reactions, such as PCR. In some embodiments, the present invention provides methods and compositions for reducing signal from background amplification products, particularly primer-dimer amplification products. In some embodiments, the reduction of signal from primer-dimer products comprises use of primers having a tag sequence. When each of the target-specific primers for PCR amplification comprises the same tag sequence, each of the strands of the resulting amplicons will comprise self-complementary portion (i.e., the tag from the primer at or near the 5' end, and the complement of the tag sequence at or near the 3' end. For long products, single-stranded portion of the adapters at each end of the ssDNA fragment may form either a self-annealing "panhandle-like" structure (amplification suppressive structure) or a DNA/primer "hybrid" structure (amplification permissive structure). The relative ratio of formation of the two structures using the subject method during PCR cycling depends on a number of factors, including the differences between the melting temperatures of the suppressive and permissive structures, the position of the complementary primer binding site within the adapter sequence, and the size of the DNA fragment to be amplified. These factors can be manipulated to achieve the desired suppression of non-target DNA during PCR amplification. In some embodiments, the formation of panhandle structures in primer-dimer products separates interactive labels, so as to reduce or eliminate detection of primer-dimer products formed in the reaction. [0009] In some embodiments the present invention comprises a method for detecting a target nucleic acid, comprising: a) contacting the sample with a polymerase; a first oligonucleotide primer comprising a 3' region complementary to a first portion of the target nucleic acid and a 5' region comprising a tag sequence; a second oligonucleotide primer comprising a 3' region comprising a sequence complementary to a second portion of the target nucleic acid, a 5' region comprising the tag sequence, and a 5' terminal region comprising a non-natural base; b) conducting a polymerase chain reaction to produce an amplified sample under conditions wherein the target nucleic acid, if present in the sample, is amplified using the first and second oligonucleotide primers to generate amplification products comprising a target amplification product having (i) a double-stranded region and (ii) a single-stranded region that comprises the non-natural base, c) contacting said amplified sample with a reporter comprising a label and a non-natural base that is complementary to the non-natural base of the single-stranded region; d) incorporating the reporter into the amplification product opposite the non-natural base of the single-stranded region; and e) detecting the incorporation of the reporter; wherein the detection of the incorporation of the reporter correlates with presence of the target nucleic acid in the sample. In some embodiments, the method further comprises the step e) of treating the amplified sample under conditions wherein non-target amplification product consisting essentially of primer-dimer product is treated to separate the strands, wherein each separated strand forms a duplex region comprising a tag sequence and a region that is complementary to the tag sequence. [0010] In some embodiments, each of the second oligonucleotide primer and said reporter comprise interactive labels. In some preferred embodiments, the interactive labels comprise a fluorophore. In some embodiments, said interactive labels comprise a quencher. [0011] In some embodiments, the step of contacting the sample with a reporter comprises contacting the sample with a reporter comprising a label and a nucleoside triphosphate of a non-natural base that is complementary to the non-natural base of the single-stranded region. In some embodiments, the step of contacting the sample with a reporter comprises contacting the sample with a reporter consisting essentially of a label and a nucleoside triphosphate of a non-natural base that is complementary to the non-natural base of the single-stranded region. [0012] In some embodiments, the incorporating step comprises incorporating the reporter into the amplification product opposite the non-natural base of the single-stranded region using a nucleic acid polymerase. In other embodiments, the incorporating step comprises incorporating the reporter into the amplification product opposite the non-natural base of the single-stranded region using a ligase. [0013] In some embodiments, the present invention comprises a kit comprising: a first oligonucleotide primer comprising a 3' region complementary to a first portion of the target nucleic acid and a 5' region comprising a tag sequence, a second oligonucleotide primer comprising a 3' region comprising a sequence complementary to a second portion of the target nucleic acid, a 5' region comprising the tag sequence, and a 5' terminal region comprising a non-natural base; and a reporter comprising a label and a non-natural base that is complementary to the non-natural base in said second oligonucleotide primer. In some embodiments the kit further comprises a third oligonucleotide primer comprising a 3' portion consisting essentially of said tag sequence. [0014] In some embodiments of the kits of the present invention, the reporter comprises an oligonucleotide comprising the non-natural base. In some embodiments, the reporter does not include any base other than the non-natural base. [0015] In some embodiments, the second region of the second oligonucleotide primer further comprises a label and the labels of the reporter and the second region of the second oligonucleotide primer comprise a pair of fluorophores where the emission of one of the fluorophores stimulates the emission of the other fluorophore. [0016] In some embodiments, the second region of the second oligonucleotide primer further comprises a label and the labels of the reporter and the second region of the second oligonucleotide primer comprise a signal generating element and a signal quenching element. DEFINITIONS [0017] To facilitate an understanding of the present invention, a number of terms and phrases are defined below: [0018] As used herein, the terms "subject" and "patient" refer to any organism, including a plant, a microorganism or an animal (e.g., a mammal such as a dog, cat, livestock, or human, or a non-mammal, such as a bird, an amphibian or a fish). [0019] As used herein, the term "kit" refers to any delivery system for delivering materials. In the context of reaction assays, such delivery systems include systems that allow for the storage, transport, or delivery of reaction reagents (e.g., oligonucleotides, enzymes, etc. in the appropriate containers) and/or supporting materials (e.g., buffers, written instructions for performing the assay etc.) from one location to another. For example, kits include one or more enclosures (e.g., boxes) containing the relevant reaction reagents and/or supporting materials. As used herein, the term "fragmented kit" refers to delivery systems comprising two or more separate containers that each contains a subportion of the total kit components. The containers may be delivered to the intended recipient together or separately. For example, a first container may contain an enzyme for use in an assay, while a second container contains oligonucleotides. The term "fragmented kit" is intended to encompass kits containing Analyte specific reagents (ASR's) regulated under section 520(e) of the Federal Food, Drug, and Cosmetic Act, but are not limited thereto. Indeed, any delivery system comprising two or more separate containers that each contains a subportion of the total kit components are included in the term "fragmented kit." In contrast, a "combined kit" refers to a delivery system containing all of the components of a reaction assay in a single container (e.g., in a single box housing each of the desired components). The term "kit" includes both fragmented and combined kits. [0020] The term "label" as used herein refers to any atom or molecule that can be used to provide a detectable (preferably quantifiable) effect, and that can be attached to a nucleic acid or protein. Labels include but are not limited to dyes; radiolabels such as .sup.32P; binding moieties such as biotin; haptens such as digoxgenin; luminogenic, phosphorescent or fluorogenic moieties; mass tags; and fluorescent dyes alone or in combination with moieties that can suppress or shift emission spectra by fluorescence resonance energy transfer (FRET). Labels may provide signals detectable by fluorescence, radioactivity, colorimetry, gravimetry, X-ray diffraction or absorption, magnetism, enzymatic activity, characteristics of mass or behavior affected by mass (e.g., MALDI time-of-flight mass spectrometry), and the like. A label may be a charged moiety (positive or negative charge) or alternatively, may be charge neutral. Labels can include or consist of nucleic acid or protein sequence, so long as the sequence comprising the label is detectable. Continue reading... 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In some embodiments, the methods can be performed entirely using computer resources, relying solely on publicly available biological sequence information. The methods of the invention can be used to generate species-specific nucleic acid microarrays ... ###  How KEYWORD MONITOR works... a FREE service from FreshPatents1. Sign up (takes 30 seconds). 2. Fill in the keywords to be monitored. 3. Each week you receive an email with patent applications related to your keywords. Start now! - Receive info on patent apps like Methods and compositions for nucleic acid analysis or other areas of interest. ### Previous Patent Application: Method to predict the response to lithium treatment Next Patent Application: Methods and computer software products for identifying transcribed regions of a genome Industry Class: Chemistry: molecular biology and microbiology ### FreshPatents.com Support Thank you for viewing the Methods and compositions for nucleic acid analysis patent info. 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