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  Dna polymerase blends and mutant dna polymerasesUSPTO Application #: 20060292578Title: Dna polymerase blends and mutant dna polymerases Abstract: A thermostable DNA polymerase composition comprising at least two DNA polymerases, one of which is substantially reduced in 5′-exonuclease activity and one of which has 5′-exonuclease activity. This polymerase may be used in methods including, but not limited to, nucleic acid synthesis, DNA sequencing, nucleic acid amplification and cDNA synthesis, (end of abstract) Agent: Invitrogen Corporation - Eugene, OR, US Inventors: Weidong Zheng, Jun E. Lee, Robert Jason Potter, David Mandelman USPTO Applicaton #: 20060292578 - 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 20060292578. Brief Patent Description - Full Patent Description - Patent Application Claims
FIELD OF THE INVENTION [0001] The present invention relates DNA polymerase blends and mutant DNA polymerases. More specifically, the invention relates to a combination of DNA polymerases which have, and which are substantially reduced in, 5-exonuclease activity. BACKGROUND OF THE INVENTION [0002] DNA polymerases synthesize formation of DNA molecules that are complementary to all or a portion of a nucleic acid template. Upon hybridization of a primer to the single-stranded template, polymerases synthesize DNA in the 5' to 3' direction, i.e., successively adding nucleotides to the 3'-hydroxyl group of the growing strand. Thus, for example, in the presence of deoxynucleoside triphosphates (dNTPs) and a primer, a new DNA molecule, complementary to the single stranded nucleic acid template, can be synthesized. Typically an RNA or DNA template is used for synthesizing a complementary DNA molecule. However, other templates, such as chimeric templates or modified nucleic acid templates are also usable for synthesizing complementary molecules of polymerized nucleic acids. A DNA-dependent DNA polymerase utilizes a DNA template and produces a DNA molecule complementary to at least a portion of the template. An RNA-dependent DNA polymerase, i.e. a reverse transcriptase, utilizes an RNA template to produce a DNA strand complementary to at least a portion of the template, i.e., a cDNA. A common application of reverse transcriptase has been to transcribe mRNA into cDNA. Some DNA polymerases have both DNA-dependent DNA polymerase activity and RNA-dependent DNA polymerase activity. [0003] In addition to a polymerase activity, DNA polymerases may possess one or more additional catalytic activities. Typically, DNA polymerases may have a 3'-5' exonuclease ("proofreading") and a 5'-3' exonuclease activity. Each of these activities has been localized to a particular region or domain of the protein. For example, when E. coli polymerase I (pol I) is cleaved into two fragments by subtilisin, the larger ("Klenow") fragment has 3'-5' exonuclease and DNA polymerase activities and the smaller fragment has 5'-3' exonuclease activity. [0004] DNA polymerases have been isolated from a variety of mesophilic and thermophilic organisms. DNA polymerases from thermophilic organisms typically have a higher optimum temperature for polymerization activity than enzymes isolated from mesophilic organisms. Thermostable DNA polymerases have been discovered in a number of thermophilic bacterial species, including, but not limited to, Thermus aquaticus (Taq), Thermus filiformis (Tfi), Thermus thermophilus (Tth), and species of the Bacillus, Thermococcus, Sulfolobus and Pyrococcus genera. In addition, thermostable DNA polymerases from a variety of other thermophiles are described in PCT WO 03/025132, the entire contents of which are incorporated herein by reference. Thermostable DNA polymerases have been exploited in numerous applications, including the polymerase chain reaction (PCR). [0005] PCR is used to amplify a target nucleic acid by denaturation of the target DNA, hybridization of oligonucleotide primers to specific sequences on opposite strands of the target DNA molecule, and subsequent extension of these primers with a DNA polymerase, usually a thermostable DNA polymerase, to generate two new strands of DNA which then serve as templates for a further round of hybridization and extension. If the polymerase is thermostable, then there is no need to add fresh polymerase after every denaturation step since heat will not have destroyed the polymerase activity. In RT-PCR, a DNA primer is hybridized to a strand of the target RNA molecule, and subsequent extension of this primer with a reverse transcriptase generates a new strand of DNA (i.e., cDNA), which can serve as a template for PCR. [0006] Thermostable DNA polymerases from Thermus aquaticus (Taq) made PCR feasible. Other thermostable polymerases having different properties (e.g., higher or lower fidelity; additional, enhanced, fewer or reduced catalytic activities; altered substrate use or preference; or different cofactor requirements) suitable for particular applications have been isolated from other organisms and/or made using recombinant DNA techniques. SUMMARY OF THE INVENTION [0007] One embodiment of the present invention is an isolated recombinant nucleic acid molecule encoding a Thermus filiformis (Tfi) DNA polymerase having a D144A point mutation, wherein said point mutation substantially reduces the 5'-exonuclease activity of said polymerase. The isolated nucleic acid molecule may further comprise an E437D point mutation. In one aspect of this embodiment, the mutant Tfi DNA polymerase is produced from one of the nucleic acid molecules described above. [0008] In another embodiment, there is provided an isolated mutant Tfi DNA polymerase having a D144A point mutation. The present invention also provides an isolated mutant Tfi DNA polymerase having D144A and E437D point mutations. Another embodiment is a composition comprising at least two thermostable DNA polymerases wherein at least one of the polymerases is substantially reduced in 5' exonuclease activity (exo-) and wherein at least one of the polymerases has 5' exonuclease activity (exo+). The polymerases may be from the same species of thermophilic bacteria. The composition may further comprise at least two of the following components: detergent, buffer salt, deoxynucleoside triphosphate (dNTP) and dideoxynucleoside triphosphate (dNTP). In another embodiment, the composition comprises a detergent, buffer salt and dNTP. In one aspect, the 5'-exo- and 5'-exo+ polymerases are combined in a ratio of between 9:1 and 1:9 (exo-:exo+). In one embodiment, the ratio is 7:3 (exo-:exo+). The present invention also provides a vector comprising any of the isolated nucleic acid molecules described above, as well as a host cell comprising the vector. The nucleic acid molecule may be operably linked to a promoter. [0009] The present invention also provides a method of synthesizing a double-stranded DNA molecule, comprising hybridizing a primer to a first DNA molecule; and incubating the DNA molecule in the presence of one or more deoxy- or dideoxyribonucleoside triphosphates and any of the DNA polymerases/compositions described above under conditions sufficient to synthesize a second DNA molecule complementary to all or a portion of the first DNA molecule. [0010] Another embodiment is a method of amplifying a double stranded DNA molecule, comprising providing a first and second primer wherein the first primer is complementary to a sequence at or near the 3'-terminus of the first strand of the DNA molecule and the second primer is complementary to a sequence at or near the 3'-terminus of the second strand of the DNA molecule; hybridizing the first primer to the first strand and the second primer to the second strand in the presence of any of the DNA polymerases/compositions described above, under conditions such that the third strand complementary to the first strand and a fourth strand complementary to the second strand are synthesized; denaturing the first and third strands and the second and fourth strands; and repeating these steps one or more times. [0011] There is also provided a kit for sequencing a DNA molecule, comprising a first container comprising any of the DNA polymerases/compositions described above; a second container comprising one or more dideoxyribonucleoside triphosphates; and a third container comprising one or more deoxyribonucleoside triphosphates. [0012] The present invention also provides a kit for amplifying a DNA molecule, comprising a first container comprising any of the DNA polymerases/compositions described above; and a second container comprising one or more deoxyribonucleoside triphosphates. [0013] Another embodiment is a method of preparing cDNA from mRNA, comprising contacting mRNA with an oligo(dT) primer or other complementary primer to form a hybrid; and contacting the hybrid with any of the DNA polymerases/compositions described above and dATP, dCTP, dGTP and dTTP, whereby a cDNA-RNA hybrid is obtained. [0014] The present invention also provides a method of preparing dsDNA from mRNA, comprising contacting mRNA with an oligo(dT) primer or other complementary primer to form a hybrid; and contacting the hybrid with any of the DNA polymerases/compositions described above, dATP, dCTP, dGTP, and dTTP, and an oligonucleotide or primer which is complementary to the a first strand cDNA, whereby dsDNA is obtained. BRIEF DESCRIPTION OF THE DRAWINGS [0015] FIG. 1 is a representative chromatogram showing BRCA exon 11 437 bp amplification products using three DNA polymerases: Tfi exo-/exo+ blend (7:3 ratio) (top line); Taq (Invitrogen) (middle line); and Taq (Promega) (bottom line). The percentage of each peak is shown. The major peak is the homoduplex peak which shows the presence of PCR induced errors. [0016] FIG. 2 is a photograph of an agarose gel showing PCR amplification products of the Rhod 1495 gene using different ratios of 5'-(exo+) and 5'-(exo-) Tfi DNA polymerases. For each reaction, 5 units of enzymes having different exo-:exo+ ratios were added. The ratios shown at the top of the gel are exo-:exo+. Ten .mu.l of each reaction was analyzed on agarose gels containing ethidium bromide. [0017] FIG. 3 is a photograph of a gel comparing the sensitivity of Tfi exo-/exo+ blend (7:3 ratio) to Taq DNA polymerase (Invitrogen). Different amounts of plasmid pBR322 or genomic K562 cell DNA fragments of about 1,000 bp were amplified and analyzed by agarose gel electrophoresis. DETAILED DESCRIPTION OF THE INVENTION [0018] The present invention relates to thermostable DNA polymerase blends, and mutant DNA polymerases, which are substantially reduced in 5'-exonuclease activity. These polymerase blends and polymerases modulate 5'-exonuclease activity to enhance PCR performance. As used herein, the term "blend" refers to at least two DNA polymerases, at least one of which is substantially reduced in 5'-exonuclease activity (referred to herein as "exo-"), and at least one of which has 5'-exonuclease activity (referred to herein as "exo+"). The exo- and exo+ polymerases may be the same or different. For example, the blend may be an exo- Tfi polymerase combined with an exo+ Tfi polymerase, or may be an exo- Tfi polymerase combined with an exo+ Taq DNA polymerase. Tfi has about 78% identity and 86% similarity at the amino acid level compared to Taq. Both Tfi and Taq DNA polymerase have 5' to 3' exonuclease and 5' to 3' polymerase activities, yet lack 3' to 5' exonuclease activity. The exo- polymerases in the exo-/exo+ blends are substantially reduced in 5'-exonuclease activity. In addition to exo+/exo- blends, also described are thermostable exo- DNA polymerases, which are reduced or substantially reduced in 5'-exo activity, particularly Tfi polymerases. Compositions, reaction mixtures, and kits containing such DNA polymerase blends, and exo- DNA polymerases, are also described herein, as are methods for nucleic acid synthesis, sequencing and amplification using these polymerases. The following terms are commonly used by those skilled in the art of molecular biology. [0019] Cloning vector. A nucleic acid molecule, for example a plasmid, cosmid or phage DNA or other DNA molecule, that is able to replicate autonomously in a host cell. A cloning vector may have one or a small number of recognition sites (e.g., recombination sites, restriction sites, topoisomerase sites, etc.) at which such DNA sequences may be manipulated in a determinable fashion without the loss of an essential biological function of the vector, and into which a nucleic acid segment of interest may be inserted in order to bring about its replication and cloning. The cloning vector may further contain a marker suitable for use in the identification of cells transformed with the cloning vector. Markers may be, for example, antibiotic resistance such as tetracycline resistance, ampicillin resistance or kanamycin resistance genes. Any other marker sequence known to those skilled in the art may be used. Continue reading... Full patent description for Dna polymerase blends and mutant dna polymerases Brief Patent Description - Full Patent Description - Patent Application Claims Click on the above for other options relating to this Dna polymerase blends and mutant dna polymerases 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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