METHODS FOR AMPLIFICATION AND SEQUENCING USING THERMOSTABLE TTHPRIMPOL
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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.
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Citations
42 Claims
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1-23. -23. (canceled)
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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. - View Dependent Claims (25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36)
- 1 and further comprises polymerase and primase activity, and
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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.
- View Dependent Claims (39, 40, 41, 42)
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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):
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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, andb. 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., andg. 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.
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Specification