Nucleic acid sequencing and mapping
First Claim
Patent Images
1. A method for sequencing nucleic acid, comprising:
- a) providing;
i) substantially double-stranded nucleic acid template,ii) a polymerase having synthetic activity and a 5'"'"'-3'"'"' exonuclease activity,iii) one or more nucleic acid precursors, andiv) one or more terminating agents;
b) mixing said polymerase, said one or more precursors, said one or more terminating agents and said template under conditions such that nucleic acid synthesis takes place for a reaction period during which said template remains substantially double-stranded and nucleic acid product is created containing said one or more terminating agents; and
c) detecting said product of said reaction under conditions whereby the nucleic acid sequence of at least a portion of said template is revealed.
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Abstract
An improvement over the standard Sanger Method for nucleic acid sequencing is described. The novel method does not require denaturation of double-stranded template; rather, sequencing can be carried out directly on the double-stranded template. Embodiments are described with and without oligonucleotide primers.
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Citations
21 Claims
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1. A method for sequencing nucleic acid, comprising:
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a) providing; i) substantially double-stranded nucleic acid template, ii) a polymerase having synthetic activity and a 5'"'"'-3'"'"' exonuclease activity, iii) one or more nucleic acid precursors, and iv) one or more terminating agents; b) mixing said polymerase, said one or more precursors, said one or more terminating agents and said template under conditions such that nucleic acid synthesis takes place for a reaction period during which said template remains substantially double-stranded and nucleic acid product is created containing said one or more terminating agents; and c) detecting said product of said reaction under conditions whereby the nucleic acid sequence of at least a portion of said template is revealed. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9)
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10. A method for sequencing nucleic acid, comprising:
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a) providing; i) substantially double-stranded nucleic acid template, ii) an endonuclease capable of specifically nicking one of the strands of said double-stranded nucleic acid template, iii) a polymerase having synthetic activity and a 5'"'"'-3'"'"' exonuclease activity, iv) one or more nucleic acid precursors, and v) one or more terminating agents; b) mixing said substantially double-stranded template with said endonuclease under conditions such that a substantially double-stranded template is produced containing a nick on one strand; c) adding a solution to said nicked template, said solution comprising said polymerase, said one or more precursors, and said one or more terminating agents, whereby said adding is carried out under conditions such that nucleic acid synthesis takes place for a reaction period during which said template remains substantially double-stranded and nucleic acid product is created containing said one or more terminating agents; and d) detecting said product of said reaction under conditions whereby the nucleic acid sequence of at least a portion of said template is revealed. - View Dependent Claims (11, 12, 13, 14, 15, 16, 17, 18, 20, 21)
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19. A method for sequencing nucleic acid, comprising:
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a) providing; i) substantially double-stranded nucleic acid template, said substantially double-stranded template comprising a single-stranded region, ii) a polymerase having synthetic activity and a 5'"'"'-3'"'"' exonuclease activity, iii) one or more nucleic acid precursors, iv) one or more terminating agents, and v) a primer capable of hybridizing to said single-stranded region of said substantially double-stranded template; b) mixing said polymerase, said one or more precursors, said one or more terminating agents, said primer and said template under conditions such that nucleic acid synthesis takes place for a reaction period during which said template remains substantially double-stranded and nucleic acid product is created containing said one or more terminating agents; and c) detecting said product of said reaction under conditions whereby the nucleic acid sequence of at least a portion of said template is revealed.
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Specification