Method of determining the nucleotide sequence of oligonucleotides and DNA molecules
DC CAFCFirst Claim
1. A method for sequencing nucleic acid, the method comprising the steps of:
- hybridizing nucleic acid to a complementary nucleic acid sequence to form one or more duplexes;
adding at least one nucleotide comprising a detectable label to said duplex, wherein said nucleotide is not a dideoxy nucleotide and wherein said nucleotide has a free 3′
hydroxyl when added to said duplex;
identifying said nucleotide comprising said detectable label; and
repeating said adding and identifying steps, thereby to determine the sequence of said nucleic acid.
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Abstract
The present invention relates to a novel method for analyzing nucleic acid sequences based on real-time detection of DNA poly-merase-catalyzed incorporation of each of the four nucleotide bases, supplied individually and serially in a microfluidic system, to a reaction cell containing a template system comprising a DNA fragment of unknown sequence and an oligonucleotide primer. Incorporation of a nucleotide base into the template system can be detected by any of a variety of methods including but not limited to fluorescence and chemiluminescence detection. Alternatively, microcalorimetic detection of the heat generated by the incorporation of a nucleotide into the extending template system using thermopile, thermistor and refractive index measurements can be used to detect extension reactions.
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Citations
8 Claims
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1. A method for sequencing nucleic acid, the method comprising the steps of:
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hybridizing nucleic acid to a complementary nucleic acid sequence to form one or more duplexes; adding at least one nucleotide comprising a detectable label to said duplex, wherein said nucleotide is not a dideoxy nucleotide and wherein said nucleotide has a free 3′
hydroxyl when added to said duplex;identifying said nucleotide comprising said detectable label; and repeating said adding and identifying steps, thereby to determine the sequence of said nucleic acid. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8)
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Specification