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  Methods, compositions, and kits for amplifying and sequencing polynucleotidesUSPTO Application #: 20060141487Title: Methods, compositions, and kits for amplifying and sequencing polynucleotides Abstract: In one aspect, there are provided methods of amplifying and sequencing a polynucleotide. In some embodiments, the method includes (a) amplifying the polynucleotide with at least one amplification primer, a processive amplification polymerase, a sequencing primer, a sequencing polymerase, deoxynucleoside triphosphates suitable for template-dependent primer extension, and one or more terminating nucleotides, the incubation being carried out at a first temperature suitable for amplifying the polynucleotide with the processive amplification polymerase; (b) incubating the product of step (a) at a second temperature suitable for forming a plurality of differently-sized extended sequencing primers with the sequencing polymerase; (c) evaluating the extended sequencing primers in order to determine the sequence of the polynucleotide. The reactions at the first and second temperatures can be carried out in a single reaction vessel. In other aspects, compositions and kits for carrying out the methods are also provided. (end of abstract) Agent: Ann M. Caviani Pease Dechert LLP - Palo Alto, CA, US Inventor: Peter Ma USPTO Applicaton #: 20060141487 - 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 20060141487. 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 application Ser. No. 60/539,465, entitled "Methods, Compositions, and Kits for Amplifying and Sequencing Polynucleotides," filed Jan. 26, 2004, the disclosure of which is incorporated herein by reference in its entirety. 2. FIELD [0002] The present disclosure relates to the field of molecular biology, and in particular, provides methods, compositions and kits for amplifying and sequencing polynucleotides. 3. INTRODUCTION [0003] Polynucleotide amplification and sequencing are fundamental technologies in molecular biology and life sciences in general. A number of methods have been developed for amplification of polynucleotides. These include the polymerase chain reaction (PCR), ligase chain reaction (LCR), self-sustained sequence replication (3SR), polynucleotide sequence based amplification (NASBA), strand displacement amplification (SDA), amplification with Q.beta. replicase (Birkenmeyer and Mushahwar, J. Virological Methods, 35:117-126 (1991); Landegren, Trends Genetics 9:199-202 (1993)), and multiple strand displacement amplification (MSDA) (U.S. Pat. No. 6,280,949). Polynucleotide amplification procedures routinely are conducted in aqueous solution. The amplification product can be viscous and difficult to accurately pipette. In order to carry out subsequent analyses, such as sequencing, the product of the amplification reaction can be subjected to manipulative procedures such as liquid transfer, dilution, centrifugation, purification, filtration and addition of enzymes and other reagents. [0004] DNA sequencing involves the generation of four populations of single-stranded DNA fragments, having one defined terminus and one variable terminus. The variable terminus terminates at a specific given nucleotide base (either guanine (G), adenine (A), thymine (T), or cytosine (C)). The four different sets of fragments are each separated on the basis of their length, such as on a high resolution polyacrylamide gel; each band on the gel corresponds colinearly to a specific nucleotide in the DNA sequence, thus identifying the positions in the sequence of the given nucleotide base. One method of DNA sequencing is dideoxy sequencing, and involves the enzymatic synthesis of a DNA strand. Typically, four separate syntheses are run, each reaction being caused to terminate at a specific base (G, A, T or C) via incorporation of the appropriate chain terminating dideoxynucleotide. Dideoxy sequencing (Sanger et al., 1997, Proc. Nat. Acad. Sci. 74:5463) requires a single-stranded template to which the primer can anneal. Single-stranded templates can be easily generated using specialized cloning vectors such as YAC, cosmids, plasmids and M13 vectors (Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press; and Ausubel et al., eds., 1996, Current Protocols in Molecular Biology, John Wiley & Sons, pp. 7.1.2-7.1.6). Dideoxy sequencing can also be readily carried out using double-stranded DNA if it is first denatured with alkali or heat. [0005] Automated robotic systems are available for high-throughput sample processing. However, each additional manipulative step slows down the overall analysis, increases the risk of contamination and increases the risk of contamination. There is a need for methods and compositions that eliminate fluid transfer steps and other manipulations that are commonly required in proceeding from amplification to sequencing. 4. SUMMARY [0006] In a general aspect, the present teachings concerns methods, compositions, and kits useful for amplifying and sequencing at least a region of a polynucleotide. The method includes an amplification step and a sequencing step. These steps can be carried out in the same reaction vessel or well, thus avoiding liquid transfers and other sample manipulations. [0007] In some embodiments, the method includes incubating a target polynucleotide with a composition comprising an amplification primer, a processive amplification polymerase, a sequencing primer, a sequencing polymerase, deoxynucleoside triphosphates suitable for template-dependent primer extension, and a terminating nucleotide. The incubation can be carried out at a first temperature which can be suitable for amplifying the nucleic acid with the processive amplification polymerase. The amplification product of the incubation at the first temperature can be incubated at a second temperature which can be suitable for forming a plurality of differently-sized extended sequencing primers with the sequencing polymerase. Thus, a temperature switch can be used to alter the annealing conditions of the incubation. In some embodiments, the incubations at the first and second temperatures are repeated in four separate incubations in the presence of a terminating nucleotide suitable for terminating primer extension at A, C, G/I, or T/U, respectively. The sequencing primer can be labeled with a detectable label. In some embodiments, the incubations at the first and second temperatures include four different terminating nucleotides, each of which terminates template-dependent primer extension at a different template nucleotide. Each of the four different terminating nucleotide can be labeled with a different, distinguishable label. [0008] In a general feature of the above methods, the second temperature can be selected such that the amplification primer does not substantially hybridize to the amplification products generated at the first temperature, but only the sequencing primer substantially hybridizes to these amplification products. At the second temperature, extension from the sequencing primer can be initiated at the site where the sequencing primer anneals and continues until termination occurs by incorporation of a terminating nucleotide that will not support continued DNA elongation. In some embodiments, a plurality of amplification primers can be used during the incubation at the first temperature. The amplification primer(s), and sequencing primer, are typically designed such that the T.sub.m value of the amplification primer(s) is(are) lower than the T.sub.m value of the sequencing primer under the selected reaction conditions. [0009] The method can be performed using a target polynucleotide obtained by an isolation technique. Alternatively, the method can be performed directly on a sample, such as a colony or plaque sample, or an aliquot of liquid medium containing a bacterial colony or culture. [0010] Extended sequencing primers can be evaluated to determine their sequence using various standard techniques including size separation and detection methods. [0011] Other aspects disclosed herein involve compositions for use in amplifying and sequencing a polynucleotide. In some embodiments, a composition includes at least one amplification primer and a sequencing primer. In some embodiments, a composition includes a processive polymerase and a sequencing polymerase. In some embodiments, a composition can include one or more of the following: deoxynucleoside triphosphates suitable for template-dependent primer extension, and at least one terminating nucleotide. [0012] Another aspect concerns kits that can be used to carrying out the method. In some embodiments, the kits can include the following: at least one amplification primer; a sequencing primer; deoxynucleoside triphosphates suitable for template-dependent primer extension; at least one terminating nucleotide; a processive polymerase; a sequencing polymerase; one or more reagents; and instructions for carrying out the method. [0013] The novel methods, compositions and kits provided herein are based in part on the surprising discovery by Applicants that terminating nucleotides used conventionally in polynucleotide sequencing do not substantially interfere with the amplification by processive polymerases. The amplification reaction can be carried out in the presence of reagents required for a subsequent sequencing reaction, which can be performed at an elevated temperature, thereby eliminating the need to provide separate reaction mixtures and/or vessels for carrying out both polynucleotide amplification and sequencing reactions. [0014] The method, compositions, and kits can be used in a wide variety of applications such as genetic studies, forensics and diagnostics. These and other features of the present teachings are set fourth below. 5. BRIEF DESCRIPTION OF THE DRAWINGS [0015] 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. [0016] FIG. 1 illustrates sequencing data obtained in which an amplification and sequencing method was performed directly on a bacterial culture sample. [0017] FIG. 2 illustrates sequencing data obtained in which an amplification and sequencing method was performed directly on a bacterial colony sample. [0018] FIG. 3 illustrates sequencing data obtained in which an amplification and sequencing method was performed using plasmid DNA. 6. DESCRIPTION OF THE VARIOUS EMBODIMENTS Continue reading... Full patent description for Methods, compositions, and kits for amplifying and sequencing polynucleotides Brief Patent Description - Full Patent Description - Patent Application Claims Click on the above for other options relating to this Methods, compositions, and kits for amplifying and sequencing polynucleotides 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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