Modified heat-resistant dna polymerase

Abstract

The present invention provides DNA polymerases that are highly resistant to inhibitors, and that can shorten the entire nucleic acid amplification reaction time by shortening the reverse transcription reaction time in a nucleic acid amplification method, in particular, in PCR or RT-PCR. The DNA polymerase is characterized by having reverse transcription activity, and comprising at least one amino acid modification at position 509 or 744 in SEQ ID NO: 1 or 2. In particular, the amino acid modification at position 509 or 744 in SEQ ID NO: 1 or 2 is substitution with histidine, lysine, or arginine.

1 . A DNA polymerase having reverse transcription activity and having 90% or more identity to the amino acid sequence of SEQ ID NO: 1, wherein the DNA polymerase comprises an amino acid modification at at least one position selected from the group consisting of position 509 and position 744. 
     
     
         2 . The DNA polymerase according to  claim 1 , wherein the DNA polymerase has 96% or more identity to the amino acid sequence of SEQ ID NO: 1. 
     
     
         3 . A DNA polymerase having reverse transcription activity and having the amino acid sequence of SEQ ID NO: 1, wherein the DNA polymerase comprises an amino acid modification at at least one position selected from the group consisting of position 509 and position 744. 
     
     
         4 . DNA polymerase having reverse transcription activity and having 90% or more identity to the amino acid sequence of SEQ ID NO: 2, wherein the DNA polymerase comprises an amino acid modification at at least one position selected from the group consisting of position 509 and position 744. 
     
     
         5 . The DNA polymerase according to  claim 4 , wherein the DNA polymerase has 96% or more identity to the amino acid sequence of SEQ ID NO:. 
     
     
         6 . DNA polymerase having reverse transcription activity and having the amino acid sequence of SEQ ID NO: 2, wherein the DNA polymerase comprises an amino acid modification at at least one position selected from the group consisting of position 509 and position 744. 
     
     
         7 . The DNA polymerase according to  claim 1 , wherein the amino acid modification at position 509 or 744 of SEQ ID NO: 1 is substitution with an amino acid selected from the group consisting of histidine, lysine, and arginine. 
     
     
         8 . The DNA polymerase according to  claim 7 , wherein the amino acid modification at position 509 of SEQ ID NO: 1 is substitution with arginine. 
     
     
         9 . The DNA polymerase according to  claim 1 , with which a reverse transcription reaction is completed in 5 minutes or less. 
     
     
         10 . The DNA polymerase according to  claim 9 , with which a reverse transcription reaction is completed in 1 minute or less. 
     
     
         11 . The DNA polymerase according to  claim 1 , wherein the extension time per kb is 30 seconds or less. 
     
     
         12 . A nucleic acid amplification method comprising amplifying a biological sample that is not purified, using the DNA polymerase of  claim 1 . 
     
     
         13 . The nucleic acid amplification method according to  claim 12 , wherein the biological sample is at least one sample selected from the group consisting of blood-derived samples, saliva, cerebrospinal fluid, urine, and milk. 
     
     
         14 . The DNA polymerase according to  claim 2 , wherein the amino acid modification at position 509 or 744 of SEQ ID NO: 1 is substitution with an amino acid selected from the group consisting of histidine, lysine, and arginine. 
     
     
         15 . The DNA polymerase according to  claim 3 , wherein the amino acid modification at position 509 or 744 of SEQ ID NO: 1 is substitution with an amino acid selected from the group consisting of histidine, lysine, and arginine. 
     
     
         16 . The DNA polymerase according to  claim 15 , wherein the amino acid modification at position 509 of SEQ ID NO: 1 is substitution with arginine. 
     
     
         17 . The DNA polymerase according to  claim 4 , wherein the amino acid modification at position 509 or 744 of SEQ ID NO: 2 is substitution with an amino acid selected from the group consisting of histidine, lysine, and arginine. 
     
     
         18 . The DNA polymerase according to  claim 5 , wherein the amino acid modification at position 509 or 744 of SEQ ID NO: 2 is substitution with an amino acid selected from the group consisting of histidine, lysine, and arginine. 
     
     
         19 . The DNA polymerase according to  claim 6 , wherein the amino acid modification at position 509 or 744 of SEQ ID NO: 2 is substitution with an amino acid selected from the group consisting of histidine, lysine, and arginine. 
     
     
         20 . The DNA polymerase according to  claim 19 , wherein the amino acid modification at position 509 of SEQ ID NO: 2 is substitution with arginine.