DNA sequencing by stepwise ligation and cleavage
First Claim
1. A method for determining a nucleotide sequence of a polynucleotide, the method comprising the steps of:
- (a) ligating a probe to an end of a polynucleotide, the probe having a nuclease recognition site of a nuclease whose cleavage site is separate from its recognition site, and the polynucleotide having been replicated in the presence of 5-methydeoxylcytidine triphosphate;
(b) identifying one or more nucleotides at the end of the polynucleotide by the identity of the probe ligated thereto or by extending a strand of the polynucleotide or probe;
(c) cleaving the polynucleotide with a nuclease recognizing the nuclease recognition site of the probe such that the polynucleotide is shortened by one or more nucleotides; and
(d) repeating said steps (a) through (c) until said nucleotide sequence of the polynucleotide is determined.
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Accused Products
Abstract
The invention provides a method of nucleic acid sequence analysis based on repeated cycles of ligation to and cleavage of probes at the terminus of a target polynucleotide. At each such cycle one or more terminal nucleotides are identified and one or more nucleotides are removed from the end of the target polynucleotide, such that further cycles of ligation and cleavage can take place. At each cycle the target sequence is shortened by one or more nucleotides until the nucleotide sequence of the target polynucleotide is determined. The method obviates electrophoretic separation of similarly sized DNA fragments and eliminates the difficulties associated with the detection and analysis of spatially overlapping bands of DNA fragments in a gel, or like medium. The invention further obviates the need to generate DNA fragments from long single stranded templates with a DNA polymerase.
328 Citations
28 Claims
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1. A method for determining a nucleotide sequence of a polynucleotide, the method comprising the steps of:
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(a) ligating a probe to an end of a polynucleotide, the probe having a nuclease recognition site of a nuclease whose cleavage site is separate from its recognition site, and the polynucleotide having been replicated in the presence of 5-methydeoxylcytidine triphosphate; (b) identifying one or more nucleotides at the end of the polynucleotide by the identity of the probe ligated thereto or by extending a strand of the polynucleotide or probe; (c) cleaving the polynucleotide with a nuclease recognizing the nuclease recognition site of the probe such that the polynucleotide is shortened by one or more nucleotides; and (d) repeating said steps (a) through (c) until said nucleotide sequence of the polynucleotide is determined. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13)
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14. A method for determining a nucleotide sequence of a polynucleotide, the method comprising the steps of:
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(a) ligating a probe to an end of a polynucleotide, the probe having a nuclease recognition site of a nuclease whose cleavage site is separate from its recognition site; (b) identifying a plurality of nucleotides at the end of the polynucleotide by the identity of the probe ligated thereto or by extending a strand of the polynucleotide or probe; (c) cleaving the polynucleotide with a nuclease recognizing the nuclease recognition site of the probe such that the polynucleotide is shortened by one or more nucleotides; and (d) repeating said steps (a) through (c) until said nucleotide sequence of the polynucleotide is determined. - View Dependent Claims (15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28)
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Specification