DNA sequencing by parallel oligonucleotide extensions
DCFirst Claim
1. A method for determining a sequence of nucleotides in a target polynucleotide, the method comprising the steps of:
- (a) providing a probe-target duplex comprising an initializing oligonucleotide probe hybridized to a target polynucleotide, said probe having an extendable probe terminus;
(b) ligating an extension oligonucleotide probe to said extendable probe terminus, to form an extended duplex containing an extended oligonucleotide probe;
(c) identifying, in the extended duplex, at least one nucleotide in the target polynucleotide that is either (1) complementary to the just-ligated extension probe or (2) a nucleotide residue in the target polynucleotide which is immediately downstream of the extended oligonucleotide probe;
(d) generating an extendable probe terminus on the extended probe, if an extendable probe terminus is not already present, such that the terminus generated is different from the terminus to which the last extension probe was ligated; and
(e) repeating steps (b), (c) and (d) until a sequence of nucleotides in the target polynucleotide is determined.
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Abstract
Method and compositions are provided for analyzing nucleic acid sequences based on repeated cycles of duplex extension along a single stranded template. Preferably, such extension starts from a duplex formed between an initializing oligonucleotide and the template. The initializing oligonucleotide is extended in an initial extension cycle by ligating an oligonucleotide probe to its end to form an extended duplex. The extended duplex is then repeatedly extended by subsequent cycles of ligation. During each cycle, the identity of one or more nucleotides in the template is determined by a label on, or associated with, a successfully ligated oligonucleotide probe. Preferably, the oligonucleotide probe has a blocking moiety, e.g. a chain-terminating nucleotide, in a terminal position so that only a single extension of the extended duplex takes place in a single cycle. The duplex is further extended in subsequent cycles by removing the blocking moiety and regenerating an extendable terminus. The invention provides a method of sequencing nucleic acids which obviates electrophoretic separation of similarly sized DNA fragments, and which eliminates the difficulties associated with the detection and analysis of spacially overlapping bands of DNA fragments in a gel or like medium. The invention also obviates the need to generate DNA fragments from long single stranded templates with a DNA polymerase.
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Citations
19 Claims
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1. A method for determining a sequence of nucleotides in a target polynucleotide, the method comprising the steps of:
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(a) providing a probe-target duplex comprising an initializing oligonucleotide probe hybridized to a target polynucleotide, said probe having an extendable probe terminus; (b) ligating an extension oligonucleotide probe to said extendable probe terminus, to form an extended duplex containing an extended oligonucleotide probe; (c) identifying, in the extended duplex, at least one nucleotide in the target polynucleotide that is either (1) complementary to the just-ligated extension probe or (2) a nucleotide residue in the target polynucleotide which is immediately downstream of the extended oligonucleotide probe; (d) generating an extendable probe terminus on the extended probe, if an extendable probe terminus is not already present, such that the terminus generated is different from the terminus to which the last extension probe was ligated; and (e) repeating steps (b), (c) and (d) until a sequence of nucleotides in the target polynucleotide is determined. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19)
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Specification