METHODS FOR AMPLIFICATION AND SEQUENCING USING THERMOSTABLE TTHPRIMPOL

• US 20160040143A1
• Filed: 03/14/2014
• Published: 02/11/2016
• Est. Priority Date: 03/15/2013
• Status: Active Grant

• Current Assignee: 4basebio SL
• Original Assignee: Sygnis Biotech S.L.U.
• Inventors: PICHER, Angel J., BLANCO, Luis
• Granted Patent: US 11,299,718 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: C12N 15/102 Mutagenizing nucleic acids
• CPC Class Codes: C12N 9/1252 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2521/101 DNA polymerase
• CPC Class Codes: C12Q 2525/117 incorporating modified base
• CPC Class Codes: C12Q 2525/121 incorporating both deoxyribonucleotides and ribonucleotides
• CPC Class Codes: C12Q 2531/113 PCR
• CPC Class Codes: C12Y 207/07007 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase

Abstract

The present invention is directed to methods for replicating, amplifying, and sequencing of nucleic acids using the thermostable, bifunctional replicase “TthPrimPol” from Thermus thermophilus HB27. The TthPrimPol enzyme is extremely tolerant to alterations of the nucleotides of the template nucleic acid. Therefore, in one aspect the invention discloses methods for replicating, amplifying and sequencing of damaged polynucleotide templates.

Claims

1-23. -23. (canceled)

24. A method for replicating,

amplifying or sequencing of DNA in the absence of a primer comprising the following steps

a) providing

i) a polymerase, or

ii) a polymerase conjugate,

wherein the polymerase, or the polymerase moiety of the polymerase conjugate having a sequence that is at least 70% identical to SEQ ID NO:

1 and further comprises polymerase and primase activity, and

b) providing a template nucleic acid, and

c) providing nucleotides and/or nucleotide derivatives for incorporation in a complementary strand of nucleic acids, and

d) providing a suitable buffer,

e) providing a second polymerase, and

f) contacting the materials of steps a-e for a suitable amount of time.

25. The method of claim 24, wherein the second polymerase provided in step e) is selected from the group consisting of prokaryotic family A polymerases and prokaryotic family B polymerases.

26. The method according to claim 25, wherein the second polymerase is a thermostable polymerase, preferably is selected from the group consisting of Taq polymerase, Tth polymerase, Pfu polymerase, Vent polymerase.

27. The method of claim 24, wherein the second polymerase provided in step e) is a Phi29 type DNA polymerase or a conjugate thereof.

28. The method according to claim 24, wherein the polymerase of a) i), or the polymerase moiety of the polymerase conjugate of a) ii) have a sequence that is at least 80% identical to SEQ ID NO:

1

29. The method according to claim 24, wherein the polymerase of a) i), or the polymerase moiety of the polymerase conjugate of a) ii) have a sequence that is at least 90% identical to SEQ ID NO:

1

30. The method according to claim 24, wherein the polymerase of a) i) or the polymerase moiety of the polymerase conjugate of a) ii) have the sequence of SEQ ID NO:

1

31. The method according to claim 24, wherein the nucleic acid of step b) is damaged.

32. The method according to claim 31, wherein the damaged nucleic acid is characterized by:

a) comprising single strand or double strand breaches, and/or

b) comprising chemically modified nucleotides, preferably selected from the group of oxidized nucleotides and/or deaminated nucleotides, most preferably selected from the group consisting of 7,8-dihydro-8-oxoadenine, 7,8-dihydro-8-oxoguanine, thymine glycol, 5-hydroxycytisine 5-hydroxyuracil and/or

c) being derived from formaldehyde or paraformaldehyde embedded tissue.

33. The method according to claim 24, wherein at least one species of nucleotide derivatives of step c) is a chemically modified nucleotide.

34. The method of claim 33, wherein the nucleotide derivative of step c) comprises at least one species of nucleotide derivatives selected from the group consisting of:

a) oxidized nucleotide derivative, and/or

b) deaminated nucleotides,

c) derivatives comprising at least one sterically demanding side group.

35. The method according to claim 24, wherein the template nucleic acid of step b;

derives from less than 100 cells;

preferably from a single cell.

36. The method according to claim 24, wherein the amplification of contaminant DNA is suppressed.

37. A polymerase or polymerase conjugate, wherein the polymerase, or the polymerase moiety of the polymerase conjugate having a sequence that is at least 70% identical to SEQ ID NO:

1 and further comprises polymerase and primase activity.

39. A method for random mutagenesis, comprising the steps

a) contacting a nucleic acid with an oxidative compound, whereby said contacting leads to the oxidation of bases of the nucleic acid, and

b) replicating the oxygenated nucleic acid with a polymerase or a polymerase conjugate as defined in claim 37.

40. A kit for amplifying a DNA molecule, that is storable under suitable conditions for more than 20 months comprising:

a) a first container comprising a polymerase as defined in claim 37,

b) a second container comprising one or more deoxyribonucleoside triphosphates.

41. A kit for amplifying a RNA molecule, that is storable under suitable conditions for more than 20 months, comprising:

a) a first container comprising a polymerase as defined in claim 37,

b) a second container comprising one or more ribonucleoside triphosphates

42. A kit for sequencing a nucleic acid molecule, that is storable under suitable conditions for more than 20 months, comprising:

a) a first container comprising a polymerase as defined in claim 37,

b) a second container comprising one or more dideoxyribonucleoside triphosphate,

c) a third container comprising one or more deoxyribonucleoside triphosphates.

38. A method for detecting modifications, in particular for detecting products of oxidation processes, in a DNA template, comprising DNA replication according to steps a)-f):

a. providing

i. a polymerase, or

ii. a polymerase conjugate,

wherein the polymerase, or the polymerase moiety of the polymerase conjugate having a sequence that is at least 70% identical to SEQ ID NO;

1 and further comprises polymerase and primase activity, and

b. providing a template nucleic acid, and

c. providing nucleotides and/or nucleotide derivatives for incorporation in a complementary strand of nucleic acids, and

d. providing a suitable buffer, and

e. optionally providing one or more primers, and

f. contacting the materials of steps a-e for a suitable amount of time, optionally at high temperature, preferably at temperatures over 40°

C. most preferred over 50°

C., and

g. calculating from multiple sequence reads the relative percentage of incorporated nucleotides for each nucleotide position over all sequence reads of step a, and

h. correlating the overall percentage of incorporated nucleotides with the original nature of the DNA modification in the template.