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  Method for nucleic acid isolation and amplificationUSPTO Application #: 20060040300Title: Method for nucleic acid isolation and amplification Abstract: The present invention provides methods and compositions for sequence-specific isolation of polynucleotide molecules from nucleic acid populations and subsequent amplification of isolated polynucleotide molecules or fragments thereof. (end of abstract) Agent: Seed Intellectual Property Law Group PLLC - Seattle, WA, US Inventors: Johannes Dapprich, Nancy Murphy, Christian Korfhage USPTO Applicaton #: 20060040300 - 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 20060040300. Brief Patent Description - Full Patent Description - Patent Application Claims
CROSS REFERENCE TO RELATED APPLICATION [0001] This application claims the benefit of U.S. Provisional Patent Application No. 60/599,903 filed Aug. 9, 2004, which is incorporated herein by reference in its entirety. BACKGROUND OF THE INVENTION [0003] 1. Field of the Invention [0004] The invention relates generally to methods and compositions for isolating and amplifying nucleic acid molecules. [0005] 2. Description of the Related Art [0006] One major area of current clinical research is the correlation of an individual's genetic profile to a susceptibility to disease and/or response to drug therapy. This area of research, which has been labeled pharmacogenomics, offers a strategy for targeting drugs to individuals, and for elucidating genetic predispositions and risks. In addition, pharmacogenomics provides for the possibility for an improved drug discovery process based on a better understanding of the molecular bases of complex diseases. [0007] Identification of an individual's genetic profile can require the identification and amplification of particular nucleic acid sequences in the individual's genome. These particular nucleic acid sequences can include those that differ by one or a few nucleotides among individuals in the same species. For example, single-nucleotide polymorphisms (SNPS) are common variations in the DNA of individuals that are used to track inherited genetic patterns. [0008] Current methods for isolating, amplifying and identifying nucleic acid polymorphisms can be labor-intensive, expensive, and not sensitive. BRIEF SUMMARY OF THE INVENTION [0009] The present invention provides methods and compositions for isolating and amplifying nucleic acid molecules. A polynucleotide molecule of interest may be first isolated from other nucleic acid molecules in a nucleic acid population based on a specific sequence in the polynucleotide molecule and then isothermally amplified. In certain embodiments, the present invention allows for the isolation of relatively long polynucleotide molecules (e.g., about 50 kb or longer) and subsequent amplification of the isolated molecules or fragments thereof. The amplified polynucleotide molecules or fragments thereof may be further analyzed. [0010] In one aspect, the present invention provides a method for amplifying a polynucleotide molecule of interest or a fragment thereof, comprising: (a) isolating a polynucleotide molecule from a nucleic acid population using an immobilizable separation group to provide an isolated polynucleotide molecule, and (b) isothermally amplifying the isolated polynucleotide molecule or a fragment thereof. [0011] In certain embodiments, (A) the nucleic acid population comprises the polynucleotide molecule of interest, (B) one strand of the polynucleotide molecule comprises a target nucleic acid sequence and a distinguishing element, (C) the target nucleic acid sequence is within 100 nucleotides of the distinguishing element in the one strand of the polynucleotide molecules, and (D) step (a) comprises: (i) contacting the nucleic acid population with a targeting element that binds specifically to the target nucleic acid sequence in the polynucleotide molecule, (ii) selectively attaching the immobilizable separation group to the targeting element bound to the target nucleic acid sequence in the polynucleotide molecule to form a targeting element-separation group complex, (iii) immobilizing to a substrate via the separation group the targeting element-separation group complex to which the target nucleic acid sequence in the polynucleotide molecule is bound, and (iv) removing the immobilized targeting element-separation group complex to which the target nucleic acid sequence in the polynucleotide molecule is bound, thereby isolating the polynucleotide molecule from the nucleic acid population. [0012] In certain embodiments, (1) the targeting element comprises an oligonucleotide, (2) the separation group comprises an immobilizable nucleotide, and (3) the separation group is attached to the targeting element by extending the oligonucleotide in the presence of the immobilizable nucleotide, thereby forming an extension product that comprises the immobilizable nucleotide. [0013] In certain embodiments, (4) the 3' terminus of the oligonucleotide is complementary to the distinguishing element or a portion thereof in the polynucleotide molecule, (5) the immobilizable nucleotide is non-terminating, and (6) the extension product comprises multiple separation groups. [0014] In certain other embodiments, (4) the target nucleic acid sequence is immediately 3' to the distinguishing element, and (5) the immobiliable nucleotide is terminating and complementary to the distinguishing element or a portion thereof. [0015] In certain embodiments, (A) the nucleic acid population comprises the polynucleotide molecule of interest, (B) one strand of the polynucleotide molecule comprises a target nucleic acid sequence and a distinguishing element, (C) the target nucleic acid sequence is within 100 nucleotides of the distinguishing element in the one strand of the polynucleotide molecule, and (D) step (a) comprises: (i) contacting the nucleic acid population with a targeting element-separation group complex, wherein the targeting element-separation group complex binds specifically to the target nucleic acid sequence in the polynucleotide molecule, (ii) selectively stabilizing the binding of the targeting element-separation group complex to the target nucleic acid sequence in the polynucleotide molecule, (iii) immobilizing to a substrate via the separation group the stabilized targeting element-separation group complex to which the target nucleic acid sequence in the polynucleotide molecule is bound, and (iv) removing the immobilized stabilized targeting element-separation group complex to which the target nucleic acid sequence in the polynucleotide molecule is bound, thereby isolating the polynucleotide molecule from the nucleic acid population. [0016] In certain embodiments, (1) the targeting element comprises an oligonucleotide, and (2) the 3' terminus of the oligonucleotide is complementary to the distinguishing element or a portion thereof in the polynucleotide. [0017] In certain embodiments, the selective stabilization is performed by ligation. In certain other embodiments, the selective stabilization is performed by extension of the oligonucleotide using the polynucleotide molecule as a template. [0018] In certain embodiments, step (b) is performed by strand displacement amplification. [0019] In certain embodiments, step (b) is generic amplification. In certain other embodiments, step (b) is sequence-specific amplification (e.g., locus-specific amplification). In certain other embodiments, step (b) is sequence-biased amplification (e.g., locus-biased amplification). [0020] In certain embodiments, step (b) amplifies all of the regions of the isolated polynucleotide molecule. In certain other embodiments, step (b) amplifies a particular region of the isolated polynucleotide molecule. [0021] In certain embodiments, step (b) is performed in the presence of a first set of specific primers each of which is at least substantially complementary to the particular region in the strand of the polynucleotide molecule that comprises the target nucleic acid sequence. [0022] In certain related embodiments, step (b) is performed further in the presence of a second set of specific primers each of which is at least substantially complementary to the particular region in the strand of the polynucleotide molecule that does not comprise the target nucleic acid sequence. Continue reading... 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