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  Emulsion pcr and amplicon captureRelated Patent Categories: Organic Compounds -- Part Of The Class 532-570 Series, Azo Compounds Containing Formaldehyde Reaction Product As The Coupling Component, Carbohydrates Or Derivatives, Nitrogen Containing, Dna Or Rna Fragments Or Modified Forms Thereof (e.g., Genes, Etc.), Synthesis Of Polynucleotides Or OligonucleotidesThe Patent Description & Claims data below is from USPTO Patent Application 20080076909. Brief Patent Description - Full Patent Description - Patent Application Claims
1. CROSS REFERENCE TO RELATED APPLICATION [0001] This application claims benefit under 35 U.S.C. .sctn. 119(e) to provisional application No. 60/817,905, filed Jun. 30, 2006, the disclosure of which is incorporated herein by reference. 2. INTRODUCTION [0002] Current methods for clonally amplifying target polynucleotides employ at least one amplification primer attached to a microparticle or another type of surface. The use of an amplification primer attached to a surface facilitates the clonal amplification of large numbers of target polynucleotides in parallel. However, the amplification reaction is encumbered by the use of an amplification primer attached to a surface and is not as efficient in comparison to amplification reactions performed entirely in solution. Therefore, there is a need in the art for compositions and methods that permit the efficient clonal amplification of target polynucleotides. 3. SUMMARY [0003] These and other features of the present teachings are set forth herein. [0004] This disclosure provides compositions, methods, and kits for the amplification and analysis of polynucleotides. In general, the disclosure provides methods of isolating and clonally amplifying polynucleotides to produce isolated populations of amplicons ("clonal amplicons") and the attachment of clonal amplicons to surfaces. Various methods or techniques can be used to clonally amplify isolated polynucleotides, such as, PCR, including exponential, linear, log-linear, and asymmetric PCR. Therefore, in some embodiments, clonal amplification reactions can include one or more amplification primers. [0005] In some embodiments, the methods comprise isolating a target polynucleotide in various types of reaction vessels, including but not limited to, a well of multiwell plate or a hydrophilic compartment of an inverse emulsion. In some embodiments, target polynucleotides can be clonally amplified entirely in solution using at least one amplification primer that comprises a masked or caged binding moiety. Therefore, disclosed herein are methods and compositions for clonally amplifying target polynucleotides without the use of a primer attached to a surface. However, in some embodiments a surface comprising the binding partner of the binding moiety is not present within the reaction vessel during clonal amplification. In some embodiments, a surface comprising the binding partner can be added to the reaction vessel after amplification. In the various embodiments, the amplicons that are produced can be attached to the surface by unmasking the binding moiety which, as a result, binds to its binding partner. In some embodiments, a masked binding moiety can be a caged biotin and the binding partner can be an anti-biotin antibody or an avidin (e.g., avidin, streptavidin, NEUTRAVIDIN.RTM.). In some embodiments, a masked binding moiety can be a caged fluorescein. The caged biotin or fluorescein can be uncaged or activated by various methods such as irradiation with light of an appropriate wavelength. [0006] In some embodiments, a masked binding moiety can be incorporated into amplicons during the amplification reaction. For example, a nucleobase comprising a masked binding moiety can be incorporated into an amplicon by a polymerase during amplification. In some embodiments, a masked binding moiety can be incorporated into a specific site or position of an amplicon. For example, a forward primer can comprise a first non-standard nucleobase that base pairs with a second non-standard nucleobase but does not substantially base pair with A, T, G, C or U. An example of non-standard nucleobases that preferentially base pair include but is not limited to isoC and isoG. In some embodiments, a forward amplification primer can contain at least one isoC or isoG at or near the 5' terminus of a forward amplification primer. IsoC and isoG are known in the art to substantially base pair with each other but not with other nucleobases such as A, T, G, C or U. Therefore, in the example where a forward primer comprises isoC and the triphosphate of isoG is present in the amplification reaction, extension of a reverse amplification primer results in the incorporation of isoG opposite the isoC. In embodiments, wherein the isoG comprises a masked binding moiety, the masked binding moiety is incorporated into the amplicon at the position opposite the isoC. [0007] Clonal amplicons can be analyzed by various methods. In some embodiments, the methods of analysis can be suitable for analyzing various populations of isolated clonal amplicons in parallel. The number of clonal amplicons that can be analyzed in parallel can be determined at the discretion of the practitioner and can include hundreds, thousands, hundreds of thousands, or millions of clonal amplicons. The methods of analysis include but are not limited to detection, single nucleotide polymorphism analysis, sequencing and the like. In various exemplary embodiments, sequencing can be by parallel sequencing, pyrosequencing, fluorescence in situ sequencing, or massively parallel signature sequencing. 4. BRIEF DESCRIPTION OF THE FIGURES [0008] The skilled artisan will understand that the drawings, described below, are for illustration purposes only. The drawings are not intended to limit the scope of the present teachings in any way. [0009] FIG. 1 illustrates the structure of biotin (C.sub.10H.sub.16N.sub.2O.sub.3S). [0010] FIG. 2 illustrates an embodiment of the synthesis of a caged biotin. (Pirrung et al. Bioconjug Chem. 1996 May-June; 7(3):317-21; WO0040712). [0011] FIG. 3 illustrates an embodiment of an inverse emulsion 120 comprising a hydrophobic phase 125 and a plurality of hydrophilic compartments 130A-D. Hydrophilic compartment 130A contains isolated target polynucleotide 200, multiple copies of reverse primer 80, multiple copies of forward primer 100 comprising caged biotin 101, microparticle 90 comprising streptavidin 91. [0012] FIG. 4 illustrates an embodiment of an inverse emulsion 120 comprising a hydrophobic phase 125 and a plurality of hydrophilic compartments 130A-D after clonal amplification of polynucleotide 200 by PCR to yield clonal amplicons 201 comprising caged biotin 101. Irradiation (hv (.about.350 nm)) of the emulsion yields uncaged biotin 102 which binds to streptavidin 91 on microparticle 90. [0013] FIG. 5 illustrates an embodiment of an inverse emulsion 150 comprising a plurality of hydrophilic compartments 140A-B disposed upon surface 110 comprising streptavidin 91. Clonal amplicons 201 comprising uncaged biotin 102 are bound to streptavidin 91 after clonal amplification. [0014] FIG. 6 illustrates an embodiment of clonal amplicons 300, 400, 500 attached to discrete areas of surface 110. 5. DETAILED DESCRIPTION [0015] It is to be understood that both the foregoing general description, including the drawings, and the following detailed description are exemplary and explanatory only and are not restrictive of this disclosure. In this disclosure, the use of the singular includes the plural unless specifically stated otherwise. Also, the use of "or" means "and/or" unless stated otherwise. Similarly, "comprise," "comprises," "comprising" "include," "includes," and "including" are not intended to be limiting. [0016] This disclosure provides compositions, methods, and kits for the analysis of polynucleotides. In general, the disclosure provides methods of isolating and amplifying polynucleotides. "Isolated" as used herein refers to placed or standing alone, discrete, detached, separated from others. "Isolated polynucleotide" as used herein refers to a polynucleotide that is detached or separated from other polynucleotides in a manner and under conditions suitable to yield isolated amplified sequences ("isolated amplicons"). The disclosed methods of isolating and amplifying polynucleotides to yield isolated populations of amplicons can be referred to as "clonal amplification". The methods and techniques employed in the analysis of clonal amplicons can be selected at the discretion of the practitioner and include but are not limited to detection, sequencing, resequencing, quantitation, single-nucleotide polymorphism analysis, and the like. [0017] The methods disclosed herein are suitable for analyzing complex polynucleotides and complex mixtures of polynucleotides. For example, the disclosed methods can be used to sequence an entire genome (e.g., cell, organism, or virus) or a subset of specific polynucleotides sequences. Therefore, in some embodiments, specific polynucleotide sequences of interest can be selected, clonally amplified, and analyzed to the exclusion of other polynucleotide sequences that may be present in a sample. [0018] In some embodiments, a plurality of polynucleotides can be clonally amplified and analyzed in parallel. "Parallel reaction" as used herein refers to a reaction comprising a plurality of discrete regions suitable for performing a plurality of reactions simultaneously. Virtually any number of polynucleotides can be clonally amplified in parallel. For example, in various exemplary embodiments, hundreds, thousands, hundreds of thousands, millions, and even greater numbers of polynucleotides can be analyzed in parallel by the disclosed methods. In various exemplary embodiments the numbers of polynucleotides analyzed in parallel can be at least 2, 100, 500, 1000, 10000, 50000, 100000, 300000, 500000, or 1000000, and even greater. [0019] In some embodiments, limiting dilution can be used to isolate polynucleotides in a manner that is suitable for clonal amplification. For example, a sample comprising a plurality of polynucleotides can be diluted to a concentration such that aliquots of the diluted sample that can be placed into individual reaction vessels (e.g., wells of a multi-well plate) can be predicted to comprise on average >0 and <1 polynucleotide. (U.S. Application No. 20050130173, 20060040297) Therefore, a percentage of reaction vessels can be predicted on a statistical basis (e.g., Poisson distribution) to comprise an isolated polynucleotide suitable for clonal amplification. Determining a dilution suitable for obtaining isolated polynucleotides is within the abilities of the skilled artisan. Factors to be considered include but are not limited to the polynucleotide concentration and the expected number, types, and composition of various polynucleotides that may be present in a sample. In some embodiments, a dilution suitable for obtaining isolated polynucleotides from a sample can be determined empirically. Once isolated within the reaction vessels, polynucleotides can be amplified by various methods as described below to produce clonal amplicons. Continue reading... Full patent description for Emulsion pcr and amplicon capture Brief Patent Description - Full Patent Description - Patent Application Claims Click on the above for other options relating to this Emulsion pcr and amplicon capture patent application. ###  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. 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