Modified klenow fragment and application thereof

Patent metadata

Field	Value
Publication number	US-12503689-B2
Application number	US-202017776490-A
Country	US
Kind code	B2
Filing date	Sep 10, 2020
Priority date	Nov 13, 2019
Publication date	Dec 23, 2025
Grant date	Dec 23, 2025

Abstract

Provided are a modified Klenow fragment and an application thereof; specifically provided is a modified Klenow fragment, wherein at least one position or functionally equivalent position among F762, A842, 1709 and P603 in the amino acid sequence of the modified Klenow fragment contains at least one amino acid substitution mutation. The modified Klenow fragment has higher DNA polymerase activity than a wild-type Klenow fragment and can be applied to sequencing.

First claim

What is claimed is:

1 . A modified Klenow fragment comprising at least amino acids 324-928 from E. coli DNA Polymerase 1, except that said fragment further possesses substitution mutations selected from the group consisting of: A842K, A842H, A842R, 1709K, 1709H, 1709R, P603K and combinations thereof, and optionally, further possesses a substitution at F762Y.

2 . The modified Klenow fragment according to claim 1 , wherein said fragment possesses substitution mutations selected from the group consisting of F762Y+A842K; P603K; and F762Y+A842K+P603K.

3 . The modified Klenow fragment according to claim 1 , wherein said fragment possesses a substitution mutation selected from the group consisting of A842K, A842H, and A842R.

4 . The modified Klenow fragment according to claim 1 , wherein said fragment possesses a substitution mutation(s) selected from the group consisting of: 1709K, 1709H, or 1709R: A842K+1709K: F762Y+A842K+1709K.

5 . The modified Klenow fragment according to claim 1 , wherein at least one end of the amino acid sequence of the modified Klenow fragment has an amino acid tag that is less than 12 amino acids in length.

6 . The modified Klenow fragment according to claim 5 , wherein the amino acid tag is located at the N-terminus of the amino acid sequence of the modified Klenow fragment.

7 . The modified Klenow fragment according to claim 6 , wherein the amino acid tag comprises 6-10 histidine residues.

8 . The modified Klenow fragment according to claim 6 , wherein the amino acid tag comprises GSSHHHHHHSSG (SEQ ID NO:1) or HHHHHHHHHH (SEQ ID NO:7).

9 . A method for incorporating nucleotides into DNA, comprising interaction among (a) the modified Klenow fragment according to claim 1 or a mixture of the modified Klenow fragments according to claim 1 , (b) DNA and (c) a nucleotide solution.

10 . The method according to claim 9 , wherein (c) the nucleotide solution contains Mg 2+ , or Mn 2+ , or Mg 2+ and Mn 2+ .

11 . The method according to claim 10 , wherein (a) the modified Klenow fragment possesses substitution mutation(s) selected from the group consisting of: A842K, A842H, or A842R; 1709K, 1709H or 1709R; A842K+1709K; F762Y+A842K+1709K; and F762Y+1709K: and (c) the nucleotide solution contains Mg 2+ at a concentration of 5-10 mM but no Mn 2+ .

12 . The method according to claim 10 , wherein (a) the modified Klenow fragment possesses substitution mutation(s) selected from the group consisting of: F762Y+A842K: P603K: F762Y+A842K+P603K; and A842K, A842H, or A842R: and (c) the nucleotide solution contains Mn 2+ at a concentration of 0.1-5 mM but no Mg 2+ .

13 . The method according to claim 10 , wherein (a) the modified Klenow fragment or the mixture of the modified Klenow fragments comprises substitution mutations functionally equivalent to A842K and I709K, and (c) the nucleotide solution contains Mg 2+ and Mn 2+ , wherein the Mg 2+ has a concentration of 5-10 mM, and the Mn 2+ has a concentration of 0.1-5 mM.

14 . A kit for incorporating nucleotides into DNA, comprising the modified Klenow fragment according to claim 1 .

15 . The kit according to claim 14 , wherein the kit further comprises a nucleotide solution.

16 . The kit according to claim 15 , wherein the nucleotide solution contains nucleotides labeled with a fluorescent molecule.

17 . The kit according to claim 14 , further comprising a nucleotide solution, wherein the modified Klenow fragment possesses substitution mutation(s) selected from the group consisting of: A842K, A842H or A842R: 1709K, 1709H or 1709R: A842K+1709K: F762Y+A842K+1709K; and F762Y+1709K: and wherein the nucleotide solution contains Mg 2+ at a concentration of 5-10 mM but no Mn 2+ .

18 . The kit according to claim 14 , further comprising a nucleotide solution, wherein the modified Klenow fragment possesses substitution mutation(s) selected from the group consisting of: F762Y; F762Y+A842K; P603K; F762Y+A842K+P603K; and A842K, A842H, or A842R and wherein the nucleotide solution contains Mn 2+ at a concentration of 0.1-5 mM but no Mg 2+ .

19 . The kit according to claim 14 , further comprising a nucleotide solution, wherein the modified Klenow fragment possesses the substitution mutation(s) A842K and 1709K, and wherein the nucleotide solution contains Mg 2+ at a concentration of 5-10 mM and Mn 2+ at a concentration of 0.1-5 mM Mg 2+ .

Assignees

• Genemind Biosciences Co Ltd

Inventors

• Chen Qingbin
• Chen Weiyue
• Li Hongdan
• Lin Xia
• Wang Wen
• Luo Weiwei
• Sun Lei

Classifications

• C12Y207/07007

  DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase · CPC title

• C12Q1/6869

  Methods for sequencing · CPC title

• Y02A50/30

  Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change · CPC title

• C12P19/34

  Polynucleotides, e.g. nucleic acids, oligoribonucleotides · CPC title

• C07K2319/21

  containing a His-tag · CPC title