Method of nucleic acid sequence determination
First Claim
1. A method of determining whether a test nucleotide is the next correct nucleotide comprising a base complementary to the next base in a template strand immediately downstream of a primer in a primed template nucleic acid, comprising the steps of:
- (a) contacting the primed template nucleic acid with a first reaction mixture that comprises a crippled DNA polymerase comprising either a polypeptide sequence comprising SEQ ID NO;
12 or a polypeptide sequence comprising SEQ ID NO;
14 and the test nucleotide,whereby, if the test nucleotide is the next correct nucleotide, there is formed a complex comprising the primed template nucleic acid, the crippled DNA polymerase and the test nucleotide, andwherein the crippled DNA polymerase is substantially incapable of magnesium-catalyzed phosphodiester bond formation;
(b) measuring binding of the primed template nucleic acid to the crippled DNA polymerase in the presence of the test nucleotide, without chemical incorporation of the test nucleotide into the primer of the primed template nucleic acid; and
(c) determining from the results of step (b) whether the test nucleotide is the next correct nucleotide.
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Accused Products
Abstract
Provided are sequencing-by-binding methods of detecting cognate nucleotides using a crippled DNA polymerizing enzyme that possesses the ability to bind the next correct nucleotide downstream of a primer in a template-dependent fashion, but does not possess the activity needed to promote phosphodiester bond formation. Use of the crippled DNA polymerase permits interrogation of one nucleotide at a time, without incorporation of any nucleotide. Labeled nucleotides, such as fluorescently labeled nucleotides, can be used in conjunction with the crippled DNA polymerase to establish cognate nucleotide identity in a rapid manner.
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Citations
17 Claims
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1. A method of determining whether a test nucleotide is the next correct nucleotide comprising a base complementary to the next base in a template strand immediately downstream of a primer in a primed template nucleic acid, comprising the steps of:
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(a) contacting the primed template nucleic acid with a first reaction mixture that comprises a crippled DNA polymerase comprising either a polypeptide sequence comprising SEQ ID NO;
12 or a polypeptide sequence comprising SEQ ID NO;
14 and the test nucleotide,whereby, if the test nucleotide is the next correct nucleotide, there is formed a complex comprising the primed template nucleic acid, the crippled DNA polymerase and the test nucleotide, and wherein the crippled DNA polymerase is substantially incapable of magnesium-catalyzed phosphodiester bond formation; (b) measuring binding of the primed template nucleic acid to the crippled DNA polymerase in the presence of the test nucleotide, without chemical incorporation of the test nucleotide into the primer of the primed template nucleic acid; and (c) determining from the results of step (b) whether the test nucleotide is the next correct nucleotide. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17)
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Specification