JPWO2020234200A5

JPWO2020234200A5 -

Info

Publication number: JPWO2020234200A5
Application number: JP2021568525A
Authority: JP
Publication date: 2023-05-22

Claims

Type Phi29 having an amino acid sequence at least 80%, 85%, 90% , 98 %, 99% or 99.5% sequence identity with SEQ ID NO: 1 and containing the amino acid substitutions K64R and optionally M97K DNA polymerase.

30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, having the sequence of SEQ ID NO: 2 or SEQ ID NO: 4 , or in addition to the amino acid substitutions K64R and optionally M97K; 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer amino acid substitutions, additions or deletions , a Phi29-type DNA polymerase having an amino acid sequence .

3. The Phi29-type DNA of claim 1 or 2, having improved affinity for short primers, ideally in the range of 4-6 nucleotides, compared to the wild-type Phi29 polymerase having SEQ ID NO: 1. polymerase.

3. The Phi29-type DNA polymerase of claim 1 or 2, which is a Phi29 polymerase with improved primer recognition compared to the wild-type Phi29 polymerase having SEQ ID NO:1.

5. Any one of claims 1-4 for improving affinity for short primers, ideally in the range of 4-6 nucleotides, compared to the wild-type Phi29 polymerase having SEQ ID NO: 1 Use of a Phi29-type DNA polymerase, which is a Phi29-type DNA polymerase as described in 1., preferably in randomly synthesized primer-based multiple displacement amplification or TruePrime DNA amplification.

A method for replicating, amplifying or sequencing template DNA, said DNA comprising at least (a) the DNA polymerase of any one of claims 1-4 ;
(b) a buffer;
(c) magnesium chloride,
(d) a primer; and (e) a reaction mixture comprising nucleoside triphosphates.

A kit for carrying out the method of claim 6 , comprising (a) the DNA polymerase of any one of claims 1-4 , (b) a buffer solution, and (c) magnesium chloride. , said kit.

A kit for carrying out the method according to claim 6 , the DNA polymerase according to any one of claims 1 to 4 ,
(a) a PrimPol enzyme (e.g. TthPrimPol),
(b) a random trimmer,
(c) random tetramers,
(d) random pentamers,
(e) random heptamers,
(f) random octamers,
(g) dNTPs,
(h) reaction buffer;
(i) one or more of buffers for use with any of the above elements;

An isolated nucleic acid molecule comprising a nucleotide sequence encoding the Phi29-type DNA polymerase of any one of claims 1-4 .

10. A recombinant nucleic acid comprising a transcriptional regulatory sequence operably linked to a nucleic acid encoding the Phi29-type DNA polymerase of claim 9 .

11. A recombinant cell comprising the recombinant nucleic acid of claim 10 .

(a) contacting a nucleic acid template molecule with a Phi29-type DNA polymerase of any one of claims 1-4 and sufficient reagents for primer extension; and (b) A method comprising performing primer extension.

13. The method of claim 12 , wherein the reagents sufficient for primer extension comprise oligonucleotide primers such as trimers, tetramers, pentamers, hexamers, heptamers, octamers, nonamers, or 10mers .

14. The method of claim 13 , wherein said primers are random primers.

14. The method of claim 13 , wherein the reagents sufficient for primer extension comprise a primase/polymerase (eg, TthPrimPol).

said primer extension comprises ( i ) multiple displacement amplification (“MDA”) or ( ii ) rolling circle amplification or (iii) multiple annealing and looping-based amplification cycles (MALBAC); A method according to any one of claims 12-15 .

17. The method of any one of claims 12-16 , wherein said Phi29-type DNA polymerase comprises both substitutions K64R and M97K.