DNA POLYMERASES AND MUTANTS THEREOF

• US 20120094332A1
• Filed: 12/30/2010
• Published: 04/19/2012
• Est. Priority Date: 09/14/2001
• Status: Abandoned Application

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Lee, Jun, Gerard, Gary, Potter, Robert Jason, Shandilya, Harini, Griffiths, Katherine R., Gibbs, Moreland D., Bergquist, Peter L.
• Application Number:
  US12/982,804
• Publication Number:
  US 20120094332A1
• Time in Patent Office: Days
• US Class Current: 435/91.2
• CPC Class Codes: C12N 9/1252 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
• CPC Class Codes: C12N 9/1276 RNA-directed DNA polymerase (2.7.7.49), i.e. reverse transcriptase or telomerase
• CPC Class Codes: C12N 9/14 Hydrolases (3)
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2521/101 DNA polymerase
• CPC Class Codes: C12Y 207/07007 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
• CPC Class Codes: C12Y 306/01 in phosphorus-containing anhydrides (3.6.1)

Abstract

The present invention provides polypeptides having a nucleotide polymerase activity and method of enhancing polymerase activity. The polypeptides of the present invention may possess both a DNA-dependent DNA polymerase activity and an RNA-dependent DNA polymerase activity, i.e., a reverse transcriptase activity. The polypeptides of the present invention may be used in any application including, but not limited to, DNA sequencing reactions, amplification reactions, cDNA synthesis reactions, and combined cDNA synthesis and amplification reactions, e.g., RT-PCR.

Claims

1-222. -222. (canceled)

123. An isolated nucleic acid comprising a nucleotide sequence encoding a polypeptide comprising an amino acid sequence that is at least 80% identical to at least forty contiguous amino acids in any one of SEQ ID NOS:

14-25, wherein the polypeptide has a nucleotide polymerase activity.

124. The nucleic acid according to claim 123, wherein the polypeptide encoded by the nucleic acid has both a DNA-dependent and an RNA-dependent nucleotide polymerase activity.

125. An isolated nucleic acid comprising a nucleotide sequence that hybridizes under stringent conditions to a nucleic acid comprising a sequence complementary to a sequence of any one of SEQ ID NOS:

2-13, and encodes a polypeptide having a nucleotide polymerase activity.

126. The nucleic acid according to claim 125, wherein the hybridization is performed under the following conditions:

42°

C. in 50% formamide, a first wash at 65°

C. in 2×

SSC and 1% SDS, and a second wash at 65°

C. in 0.1×

SSC.

127. The nucleic acid according to claim 126, wherein the polypeptide encoded by the nucleic acid has both a DNA-dependent and an RNA-dependent nucleotide polymerase activity.

128. A polypeptide comprising an amino acid sequence that is at least 80% identical to at least forty contiguous amino acids in any one of SEQ ID NOS:

14-25 and mutants, fragments and fragments of mutants thereof wherein the polypeptide, mutant, fragment or fragment of mutant has a nucleotide polymerase activity.

129. The polypeptide according to claim 128, wherein the polypeptide has both a DNA-dependent and an RNA-dependent nucleotide polymerase activity.

132. A kit for amplifying a DNA molecule, comprising a first container containing a polypeptide according to claim 128.

133. The kit according to claim 132, further comprising a second container containing one or more deoxyribonucleoside triphosphates.

134. A kit according to claim 132, wherein the polypeptide has at least one mutation selected from the group consisting of (1) a mutation that reduces, substantially reduces or eliminates 5′

-3′

exonuclease activity of the DNA polymerase, (2) a mutation that results in the DNA polymerase becoming non-discriminating against dideoxynucleotides, and (3) a mutation that increases thermostability of an activity of the polypeptide.

135. The kit according to claim 132, wherein the deoxyribonucleoside triphosphates are selected from the group consisting of:

DATP, dCTP, dGTP, dTTP, dITP, 7-deaza-dGTP, dUTP, [α

-S]dATP, [α

-S]dTTP, [α

-S]dGTP, and [α

-S]dCTP.

136. The kit according to claim 132, wherein the kit further comprises a container containing a pyrophosphatase.

142. A nucleic acid polymerase having an RNA-dependent DNA polymerase activity, wherein the activity occurs in the presence of magnesium, manganese or a mixture of magnesium and manganese.

143. The nucleic acid polymerase according to claim 142, wherein the polymerase further has a DNA-dependent DNA polymerase activity, the DNA-dependent DNA polymerase activity occurring under the same manganese/magnesium ratios as the RNA-dependent DNA polymerase activity.

145. A nucleic acid polymerase according to claim 142, wherein the magnesium is present at a concentration of from about 0.1 to 5.0 mM.

146. The nucleic acid polymerase of claim 142, wherein the manganese is present at a concentration of about 0 to about 2 mM.

148. The nucleic acid polymerase according to claim 145, wherein the activity occurs in the absence of manganese.

144. A nucleic acid polymerase having a DNA-dependent DNA polymerase activity, wherein the activity occurs in the presence of magnesium, manganese or a mixture of magnesium and manganese.

147. The nucleic acid polymerase according to claim 144, wherein the activity occurs in the absence of manganese.

149. A polypeptide having an RNA-dependent DNA polymerase specific activity and a DNA-dependent DNA polymerase specific activity, wherein the ratio of RNA-dependent DNA polymerase specific activity to DNA-dependent DNA polymerase specific activity is greater than about 0.01.

150. The polypeptide according to claim 149, wherein the ratio is greater than about 0.2.

151. The polypeptide according to claim 149, wherein the RNA-dependent specific activity is greater than about 500 units/mg polypeptide.

152. The polypeptide according to claim 149, wherein the DNA-dependent DNA polymerase specific activity is greater than about 10,000 units/mg polypeptide.

153. The polypeptide according to claim 149, wherein the RNA-dependent DNA polymerase activity is greater than about 2,000 units/mg polypeptide.