Helicase dependent amplification of nucleic acids
First Claim
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1. A method for exponentially and selectively amplifying a target nucleic acid in a helicase-dependent reaction, the method comprising:
- (a) providing single strand templates of the target nucleic acid to be amplified;
(b) adding oligonucleotide primers for hybridizing to the templates of step (a);
(c) synthesizing an extension product of the oligonucleotide primers which are complementary to the templates, by means of a DNA polymerase to form a duplex;
(d) contacting the duplex of step (c) with a helicase preparation for unwinding the duplex such that the helicase preparation comprises a helicase and a single strand binding protein (SSB) unless the helicase preparation comprises a thermostable helicase wherein the single strand binding protein is optional; and
(e) repeating steps (b)–
(d) to exponentially and selectively amplify the target nucleic acid in a helicase-dependent reaction such that amplification does not occur in the absence of the helicase as determined by gel electrophoresis.
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Abstract
Methods and a kit are provided for selectively and exponentially amplifying nucleic acids and include the use of a helicase preparation and a DNA polymerase such that the amplification can be performed isothermally.
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Citations
47 Claims
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1. A method for exponentially and selectively amplifying a target nucleic acid in a helicase-dependent reaction, the method comprising:
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(a) providing single strand templates of the target nucleic acid to be amplified; (b) adding oligonucleotide primers for hybridizing to the templates of step (a); (c) synthesizing an extension product of the oligonucleotide primers which are complementary to the templates, by means of a DNA polymerase to form a duplex; (d) contacting the duplex of step (c) with a helicase preparation for unwinding the duplex such that the helicase preparation comprises a helicase and a single strand binding protein (SSB) unless the helicase preparation comprises a thermostable helicase wherein the single strand binding protein is optional; and (e) repeating steps (b)–
(d) to exponentially and selectively amplify the target nucleic acid in a helicase-dependent reaction such that amplification does not occur in the absence of the helicase as determined by gel electrophoresis. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44)
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45. A method for determining whether a helicase is suited for exponentially and selectively amplifying a target nucleic acid in a helicase-dependent reaction, comprising;
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(a) preparing a helicase preparation comprising the helicase, an NTP or dNTP, a buffer, wherein the buffer has a pH in the range of about pH 6.0–
9.0, a concentration of NaCl or KCl in a concentration range of 0–
200 mM, and Tris-acetate or Tris-HCl and an accessory protein to which is added one or more single strand binding proteins unless the helicase is thermostable wherein the single strand binding protein is not required;(b) adding a target nucleic acid, oligonucleotide primers, four dNTPs and a DNA polymerase to the helicase preparation; (c) incubating the mixture at a temperature between about 20°
C. and 75°
C.; and(d) analyzing the DNA on an agarose gel to determine whether selective and exponential amplification has occurred. - View Dependent Claims (46)
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47. A method for helicase-dependent amplification of a target nucleic acid, comprising:
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(a) adding to the target nucleic acid, a helicase preparation comprising a helicase and a single strand binding protein unless the helicase is a thermostable helicase in which case the single strand binding protein is not required, a plurality of oligonucleotide primers and one or more polymerases; and (b) amplifying, by helicase-dependent amplification, the target nucleic acid wherein the amplification does not occur in the absence of the helicase as determined by gel electrophoresis.
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Specification