Helicase-dependent amplification of nucleic acids
First Claim
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1. A method for helicase-dependent amplification of a target nucleic acid, comprising:
- (a) adding to the target nucleic acid, a helicase preparation, comprising;
a superfamily 1 helicase and a single strand binding protein unless the superfamily 1 helicase is a thermostable helicase in which case the single strand binding protein is not required;
one or more oligonucleotide primer pairs; and
one or more polymerases; and
(b) amplifying the target nucleic acid wherein the amplification is dependent on the helicase preparation wherein the 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
46 Claims
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1. 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 superfamily 1 helicase and a single strand binding protein unless the superfamily 1 helicase is a thermostable helicase in which case the single strand binding protein is not required;
one or more oligonucleotide primer pairs; and
one or more polymerases; and(b) amplifying the target nucleic acid wherein the amplification is dependent on the helicase preparation wherein the 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)
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44. A method for determining whether a helicase is suited for 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 SSBs unless the helicase is thermostable wherein the SSB 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 to determine whether selective amplification has occurred, wherein the amplification does not occur in the absence of the helicase as determined by gel electrophoresis. - View Dependent Claims (45)
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46. 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) hybridizing one or more oligonucleotide primer pairs 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 an SSB unless the helicase preparation comprises a thermostable helicase wherein the SSB 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 from an ethidium bromide stained gel after electrophoresis.
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Specification
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Current AssigneeBioHelix Corp. (Quidel Corporation)
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Original AssigneeNew England Biolabs Incorporated
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InventorsXu, Yan, Vincent, Myriam, Kong, Huimin
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Primary Examiner(s)Strzelecka; Teresa E
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Application NumberUS11/796,675Publication NumberTime in Patent Office1,292 DaysField of SearchNoneUS Class Current435/6.12CPC Class CodesC12Q 1/6844 Nucleic acid amplification ...C12Q 2521/513 Winding/unwinding enzyme, e...
