US 5,599,675 A · DNA sequencing by stepwise ligation and cleavage

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.

Claims68

Dependent claims

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;
(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.

2 more elements

A method for determining a nucleotide sequence of a polynucleotide, the method comprising the steps of:

(a) providing a polynucleotide in double stranded form such that the polynucleotide is attached to a solid phase support and has a protruding strand at one end;
(b) ligating a probe to the protruding strand of the polynucleotide to form a ligated complex, the probe having an end with a complementary protruding strand to that of the polynucleotide and the probe having a type IIs endonuclease recognition site;
(c) identifying a nucleotide in the protruding strand of the polynucleotide by the identity of the ligated probe;
(d) cleaving the ligated complex with a type IIs endonuclease that recognizes the type IIs endonuclease recognition site and cleaves the ligated complex to give an augmented probe and a new protruding strand on the polynucleotide;
and
(e) repeating steps (a) through (d) until the nucleotide sequence of the polynucleotide is determined.

3 more elements

A method for determining a nucleotide sequence of a population of identical polynucleotdes, the method comprising the steps of:

(a) providing a first probe having a first nuclease recognition site of a first nuclease whose cleavage site is separate from its recognition site, the first nuclease having a reach;
(b) providing a second probe having a second nuclease recognition site of a second nuclease whose cleavage site is separate from its recognition site, the second nuclease having a reach;
(c) providing a conversion probe for converting a polynucleotide of the population from one cleavable by the first nuclease to one cleavable by the second nuclease;
(d) ligating a mixture of the first probe and the conversion probe to the polynucleotides of the population to form a subpopulation of ligated complexes comprising the conversion probe;
(e) cleaving the subpopulation of ligated complexes comprising the conversion probe with the second nuclease and determining a portion of the nucleotide sequence of the resulting polynucleotides by:
i) ligating the second probe to an end of the polynucleotides of the subpopulation;
ii) identifying one or more nucleotides at the end of the polynucleotides of the subpopulation by the identity of the probe ligated thereto or by extending a strand of the polynucleotide or the second probe;
iii) cleaving the polynucleotides of the subpopulation with the second nuclease such that the polynucleotides of the subpopulation are shortened by one or more nucleotides;
iv) repeating steps i)-iii) until the number of nucleotides cleaved from the polynucleotides of the subpopulation is equal to or greater than the reach of the first nuclease;
v) capping the ends of the polynucleotides of the subpopulation;
(f) cleaving the polynucleotides with the first nuclease;
and
(g) repeating steps (d)-(f) until the nucleotide sequence of the population of polynucleotides is determined.

10 more elements

Dependencies are inferred from the claim text.

DNA sequencing by stepwise ligation and cleavage

Life of this patent

DNA sequencing by stepwise ligation and cleavage

Life of this patent

Abstract

Claims68