US5552278A
Expired / Lapsed
US
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DNA sequencing by stepwise ligation and cleavage
Assignee: Lynx Therapeutics Inc
•
Filing: 1994-07-25
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Granted: 1996-09-03
•
Expires: 2014-07-25
Abstract
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 spacially 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.
Inventors
- 👤 Sydney Brenner
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-07 18:54
CPC Classifications
C; C12; C12Q; C12Q1/00; C12Q1/68; C12Q1/6869; C12Q1/6844; C12Q1/6862; C12Q1/6874; Y; Y02; Y02P; Y02P20/00; Y02P20/50; Y02P20/582
Claims Analysis
Total Claims: 64
Claim 1
Independent Claim
157 words
A method for determining the nucleotide sequence of a polynucleotide, the method comprising the steps of: (a) providing the polynucleotide in double stranded form such that the polynucleotide has a protruding strand at at least one end; (b) ligating a probe from a mixture of probes to an end of the polynucleotide having a protruding strand to form a ligated complex, each probe having an end with a complementary protruding strand to that of the polynucleotide and each probe having a nuclease recognition site of a nuclease whose cleavage site is separate from its recognition site; (c) identifying one or more nucleotides in the protruding strand of the polynucleotide; (d) cleaving the ligated complex with said nuclease that recognizes said nuclease recognition site and cuts 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.
Claim 2
Independent Claim
157 words
A method for determining the nucleotide sequence of a polynucleotide, the method comprising the steps of: (a) providing the polynucleotide in double stranded form such that the polynucleotide has a protruding strand at at least one end; (b) ligating a probe from a mixture of probes to an end of the polynucleotide having a protruding strand to form a ligated complex, each probe having an end with a complementary protruding strand to that of the polynucleotide and each probe having a nuclease recognition site of a nuclease whose cleavage site is separate from its recognition site; (c) identifying one or more nucleotides in the protruding strand of the polynucleotide; (d) cleaving the ligated complex with said nuclease that recognizes said nuclease recognition site and cuts 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.
Claim 3
Dependent Claim
22 words
The method of claim 1 wherein the step of providing further includes blocking the recognition sites of said nuclease on said polynucleotide.
Claim 4
Dependent Claim
28 words
The method of claim 2 wherein said nuclease is a type IIs restriction endonuclease and wherein said blocking said recognition sites includes treating said polynucleotide with a methylase.
Claim 5
Dependent Claim
17 words
The method of claim 3 wherein said step of ligating includes treating said polynucleotide with a ligase.
Claim 6
Dependent Claim
34 words
The method of claim 4 wherein said polynucleotide contains a 5'-phosphoryl group on said end having said protruding strand and said probe lacks a 5'-phosphoryl group on said end having said complementary protruding strand.
Claim 7
Dependent Claim
24 words
The method of claim 5 wherein said step of ligation includes treating said polynucleotide in succession with a ligase, a kinase, and a ligase.
Claim 8
Dependent Claim
22 words
The method of claim 1 further including the step of removing unligated probe from said ligated complex after said step of ligating.
Claim 9
Dependent Claim
31 words
The method of claim 1 wherein said step of identifying includes identifying said one or more nucleotides in said protruding strand of said polynucleotide by the identity of said ligated probe.
Claim 10
Dependent Claim
58 words
The method of claim 1 wherein said protruding strand of said polynucleotide includes a 3' recessed strand and wherein step of identifying includes extending the 3' recessed strand by a nucleic acid polymerase in the presence of at least one chain-terminating nucleoside triphosphate so that at least one chain-terminating nucleoside triphosphate is incorporated into the 3' recessed strand.
Claim 11
Dependent Claim
49 words
The method of claim 9 wherein said chain-terminating nucleoside triphosphate is a labeled dideoxynucleoside triphosphate and wherein said step of identifying includes identifying said one or more nucleotides by the label on the labeled dideoxynucleoside triphosphate incorporated into said 3' recessed strand of said polynucleotide as a labeled dideoxynucleotide.
Claim 12
Dependent Claim
24 words
The method of claim 10 further including the steps of excising said labeled dideoxynucleotide and extending said recessed strand with a nucleic acid polymerase.
Claim 13
Independent Claim
144 words
A method for determining the nucleotide sequence of a polynucleotide, the method comprising the steps of: (a) ligating a probe from a mixture of probes to an end of a polynucleotide having a protruding strand to forth a ligated complex, each probe having an end with a complementary protruding strand to that of the polynucleotide and each probe having a nuclease recognition site of a nuclease whose cleavage site is separate from its recognition site; (b) removing unligated probe from the ligated complex; (c) cleaving the ligated complex with said nuclease, said nuclease recognizing the recognition site and cleaving the ligated complex to give an augmented probe and a new protruding strand on the polynucleotide; (d) identifying one or more nucleotides in the protruding strand of the polynucleotide; and (e) repeating steps (a) through (d) until the nucleotide sequence of the polynucleotide is determined.
Claim 14
Independent Claim
144 words
A method for determining the nucleotide sequence of a polynucleotide, the method comprising the steps of: (a) ligating a probe from a mixture of probes to an end of a polynucleotide having a protruding strand to forth a ligated complex, each probe having an end with a complementary protruding strand to that of the polynucleotide and each probe having a nuclease recognition site of a nuclease whose cleavage site is separate from its recognition site; (b) removing unligated probe from the ligated complex; (c) cleaving the ligated complex with said nuclease, said nuclease recognizing the recognition site and cleaving the ligated complex to give an augmented probe and a new protruding strand on the polynucleotide; (d) identifying one or more nucleotides in the protruding strand of the polynucleotide; and (e) repeating steps (a) through (d) until the nucleotide sequence of the polynucleotide is determined.
Claim 15
Dependent Claim
21 words
The method of claim 12 further including the step of providing said polynucleotide with said recognition sites of said nuclease blocked.
Claim 16
Dependent Claim
28 words
The method of claim 13 wherein said nuclease is a type IIs restriction endonuclease and wherein said blocking said recognition sites includes treating said polynucleotide with a methylase.
Claim 17
Dependent Claim
17 words
The method of claim 14 wherein said step of ligating includes treating said polynucleotide with a ligase.
Claim 18
Dependent Claim
34 words
The method of claim 15 wherein said polynucleotide contains a 5'-phosphoryl group on said end having said protruding strand and said probe lacks a 5'-phosphoryl group on said end having said complementary protruding strand.
Claim 19
Dependent Claim
24 words
The method of claim 16 wherein said step of ligation includes treating said polynucleotide in succession with a ligase, a kinase, and a ligase.
Claim 20
Independent Claim
124 words
A method for determining the nucleotide sequence of a polynucleotide, the method comprising the steps of: (a) ligating a probe from a mixture of probes to an end of a polynucleotide having a protruding strand 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 nuclease recognition site of a nuclease whose cleavage site is separate from its recognition site; (b) identifying one or more nucleotides in the protruding strand of the polynucleotide; (c) cleaving the ligated complex with said nuclease so that the polynucleotide is shortened by one or more nucleotides; and (d) repeating steps (a) through (c) until the nucleotide sequence of said nuclease polynucleotide is determined.
Claim 21
Independent Claim
124 words
A method for determining the nucleotide sequence of a polynucleotide, the method comprising the steps of: (a) ligating a probe from a mixture of probes to an end of a polynucleotide having a protruding strand 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 nuclease recognition site of a nuclease whose cleavage site is separate from its recognition site; (b) identifying one or more nucleotides in the protruding strand of the polynucleotide; (c) cleaving the ligated complex with said nuclease so that the polynucleotide is shortened by one or more nucleotides; and (d) repeating steps (a) through (c) until the nucleotide sequence of said nuclease polynucleotide is determined.
Claim 22
Dependent Claim
21 words
The method of claim 18 further including the step of providing said polynucleotide with said recognition sites of said nuclease blocked.
Claim 23
Dependent Claim
28 words
The method of claim 19 wherein said nuclease is a type IIs restriction endonuclease and wherein said blocking said recognition sites includes treating said polynucleotide with a methylase.
Claim 24
Dependent Claim
17 words
The method of claim 20 wherein said step of ligating includes treating said polynucleotide with a ligase.
Claim 25
Dependent Claim
34 words
The method of claim 21 wherein said polynucleotide contains a 5'-phosphoryl group on said end having said protruding strand and said probe lacks a 5'-phosphoryl group on said end having said complementary protruding strand.
Claim 26
Dependent Claim
24 words
The method of claim 22 wherein said step of ligation includes treating said polynucleotide in succession with a ligase, a kinase, and a ligase.
Claim 27
Dependent Claim
33 words
The method of claim 23 wherein the identity of a single nucleotide is determined in said step of identifying and said polynucleotide is shortened by a single nucleotide in said step of cleaving.
Claim 28
Dependent Claim
22 words
The method of claim 18 further including the step of removing unligated probe from said ligated complex after said step of ligating.
Claim 29
Dependent Claim
31 words
The method of claim 18 wherein said step of identifying includes identifying said one or more nucleotides in said protruding strand of said polynucleotide by the identity of said ligated probe.
Claim 30
Dependent Claim
58 words
The method of claim 18 wherein said protruding strand of said polynucleotide includes a 3' recessed strand and wherein step of identifying includes extending the 3' recessed strand by a nucleic acid polymerase in the presence of at least one chain-terminating nucleoside triphosphate so that at least one chain-terminating nucleoside triphosphate is incorporated into the 3' recessed strand.
Claim 31
Dependent Claim
45 words
The method of claim 27 wherein said chain-terminating nucleoside triphosphate is a labeled dideoxynucleoside triphosphate and wherein said step of identifying includes identifying said one or more nucleotides by the label on the labeled dideoxynucleoside triphosphate incorporated into said 3' recessed strand of said polynucleotide.
Claim 32
Independent Claim
100 words
A method for determining the identity of a terminal nucleotide of a polynucleotide, the method comprising the steps of: (a) ligating a probe from a mixture to an end of a polynucleotide having a protruding strand 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 nuclease recognition site of a nuclease whose cleavage site is separate from its recognition site; (b) removing unligated probe from the ligated complex; and (c) identifying the terminal nucleotide of the polynucleotide by the identity of the ligated probe.
Claim 33
Independent Claim
100 words
A method for determining the identity of a terminal nucleotide of a polynucleotide, the method comprising the steps of: (a) ligating a probe from a mixture to an end of a polynucleotide having a protruding strand 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 nuclease recognition site of a nuclease whose cleavage site is separate from its recognition site; (b) removing unligated probe from the ligated complex; and (c) identifying the terminal nucleotide of the polynucleotide by the identity of the ligated probe.
Claim 34
Independent Claim
148 words
A method for determining the nucleotide sequence of a polynucleotide, the method comprising the steps of: (a) providing the polynucleotide in double stranded form such that the polynucleotide has a protruding strand at at least one end; (b) ligating a probe to an end of the polynucleotide having a protruding strand to form a ligated complex, the probe having a nuclease recognition site of a nuclease whose cleavage site is separate from its recognition site and an end with a complementary protruding strand to that of the polynucleotide; (c) identifying one or more nucleotides in the protruding strand of the polynucleotide; (d) cleaving the ligated complex with a nuclease that recognizes the recognition site and cuts 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.
Claim 35
Independent Claim
148 words
A method for determining the nucleotide sequence of a polynucleotide, the method comprising the steps of: (a) providing the polynucleotide in double stranded form such that the polynucleotide has a protruding strand at at least one end; (b) ligating a probe to an end of the polynucleotide having a protruding strand to form a ligated complex, the probe having a nuclease recognition site of a nuclease whose cleavage site is separate from its recognition site and an end with a complementary protruding strand to that of the polynucleotide; (c) identifying one or more nucleotides in the protruding strand of the polynucleotide; (d) cleaving the ligated complex with a nuclease that recognizes the recognition site and cuts 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.
Claim 36
Dependent Claim
22 words
The method of claim 30 wherein the step of providing further includes blocking the recognition sites of said nuclease on said polynucleotide.
Claim 37
Dependent Claim
28 words
The method of claim 31 wherein said nuclease is a type IIs restriction endonuclease and wherein said blocking said recognition sites includes treating said polynucleotide with a methylase.
Claim 38
Dependent Claim
17 words
The method of claim 32 wherein said step of ligating includes treating said polynucleotide with a ligase.
Claim 39
Dependent Claim
34 words
The method of claim 33 wherein said polynucleotide contains a 5'-phosphoryl group on said end having said protruding strand and said probe lacks a 5'-phosphoryl group on said end having said complementary protruding strand.
Claim 40
Dependent Claim
24 words
The method of claim 34 wherein said step of ligation includes treating said polynucleotide in succession with a ligase, a kinase, and a ligase.
Claim 41
Dependent Claim
22 words
The method of claim 30 further including the step of removing unligated probe from said ligated complex after said step of ligating.
Claim 42
Dependent Claim
31 words
The method of claim 30 wherein said step of identifying includes identifying said one or more nucleotides in said protruding strand of said polynucleotide by the identity of said ligated probe.
Claim 43
Dependent Claim
58 words
The method of claim 30 wherein said protruding strand of said polynucleotide includes a 3' recessed strand and wherein step of identifying includes extending the 3' recessed strand by a nucleic acid polymerase in the presence of at least one chain-terminating nucleoside triphosphate so that at least one chain-terminating nucleoside triphosphate is incorporated into the 3' recessed strand.
Claim 44
Dependent Claim
49 words
The method of claim 38 wherein said chain-terminating nucleoside triphosphate is a labeled dideoxynucleoside triphosphate and wherein said step of identifying includes identifying said one or more nucleotides by the label on the labeled dideoxynucleoside triphosphate incorporated into said 3' recessed strand of said polynucleotide as a labeled dideoxynucleotide.
Claim 45
Dependent Claim
24 words
The method of claim 39 further including the steps of excising said labeled dideoxynucleotide and extending said recessed strand with a nucleic acid polymerase.
Claim 46
Independent Claim
139 words
A method for determining the nucleotide sequence of a polynucleotide, the method comprising the steps of: (a) ligating a probe to an end of a polynucleotide having a protruding strand 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 nuclease recognition site of a nuclease whose cleavage site is separate from its recognition site; (b) removing unligated probe from the ligated complex; (c) cleaving the ligated complex with a nuclease, the nuclease recognizing the recognition site and cleaving the ligated complex to give an augmented probe and a new protruding strand on the polynucleotide; (d) identifying one or more nucleotides in the protruding strand of the polynucleotide; and (e) repeating steps (a) through (d) until the nucleotide sequence of the polynucleotide is determined.
Claim 47
Independent Claim
139 words
A method for determining the nucleotide sequence of a polynucleotide, the method comprising the steps of: (a) ligating a probe to an end of a polynucleotide having a protruding strand 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 nuclease recognition site of a nuclease whose cleavage site is separate from its recognition site; (b) removing unligated probe from the ligated complex; (c) cleaving the ligated complex with a nuclease, the nuclease recognizing the recognition site and cleaving the ligated complex to give an augmented probe and a new protruding strand on the polynucleotide; (d) identifying one or more nucleotides in the protruding strand of the polynucleotide; and (e) repeating steps (a) through (d) until the nucleotide sequence of the polynucleotide is determined.
Claim 48
Dependent Claim
21 words
The method of claim 41 further including the step of providing said polynucleotide with said recognition sites of said nuclease blocked.
Claim 49
Dependent Claim
28 words
The method of claim 42 wherein said nuclease is a type IIs restriction endonuclease and wherein said blocking said recognition sites includes treating said polynucleotide with a methylase.
Claim 50
Dependent Claim
17 words
The method of claim 43 wherein said step of ligating includes treating said polynucleotide with a ligase.
Claim 51
Dependent Claim
34 words
The method of claim 44 wherein said polynucleotide contains a 5'-phosphoryl group on said end having said protruding strand and said probe lacks a 5'-phosphoryl group on said end having said complementary protruding strand.
Claim 52
Dependent Claim
24 words
The method of claim 45 wherein said step of ligation includes treating said polynucleotide in succession with a ligase, a kinase, and a ligase.
Claim 53
Independent Claim
118 words
A method for determining the nucleotide sequence of a polynucleotide, the method comprising the steps of: (a) ligating a probe to an end of a polynucleotide having a protruding strand 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 nuclease recognition site of a nuclease whose cleavage site is separate from its recognition site; (b) identifying one or more nucleotides in the protruding strand of the polynucleotide; (c) cleaving the ligated complex with said nuclease so that the polynucleotide is shortened by one or more nucleotides; and (d) repeating steps (a) through (c) until the nucleotide sequence of the polynucleotide is determined.
Claim 54
Independent Claim
118 words
A method for determining the nucleotide sequence of a polynucleotide, the method comprising the steps of: (a) ligating a probe to an end of a polynucleotide having a protruding strand 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 nuclease recognition site of a nuclease whose cleavage site is separate from its recognition site; (b) identifying one or more nucleotides in the protruding strand of the polynucleotide; (c) cleaving the ligated complex with said nuclease so that the polynucleotide is shortened by one or more nucleotides; and (d) repeating steps (a) through (c) until the nucleotide sequence of the polynucleotide is determined.
Claim 55
Dependent Claim
21 words
The method of claim 47 further including the step of providing said polynucleotide with said recognition sites of said nuclease blocked.
Claim 56
Dependent Claim
28 words
The method of claim 48 wherein said nuclease is a type IIs restriction endonuclease and wherein said blocking said recognition sites includes treating said polynucleotide with a methylase.
Claim 57
Dependent Claim
17 words
The method of claim 49 wherein said step of ligating includes treating said polynucleotide with a ligase.
Claim 58
Dependent Claim
34 words
The method of claim 50 wherein said polynucleotide contains a 5'-phosphoryl group on said end having said protruding strand and said probe lacks a 5'-phosphoryl group on said end having said complementary protruding strand.
Claim 59
Dependent Claim
24 words
The method of claim 51 wherein said step of ligation includes treating said polynucleotide in succession with a ligase, a kinase, and a ligase.
Claim 60
Dependent Claim
33 words
The method of claim 52 wherein the identity of a single nucleotide is determined in said step of identifying and said polynucleotide is shortened by a single nucleotide in said step of cleaving.
Claim 61
Dependent Claim
22 words
The method of claim 53 further including the step of removing unligated probe from said ligated complex after said step of ligating.
Claim 62
Dependent Claim
31 words
The method of claim 47 wherein said step of indentifying includes identifying said one or more nucleotides in said protruding strand of said polynucleotide by the identity of said ligated probe.
Claim 63
Dependent Claim
58 words
The method of claim 47 wherein said protruding strand of said polynucleotide includes a 3' recessed strand and wherein step of identifying includes extending the 3' recessed strand by a nucleic acid polymerase in the presence of at least one chain-terminating nucleoside triphosphate so that at least one chain-terminating nucleoside triphosphate is incorporated into the 3' recessed strand.
Claim 64
Dependent Claim
45 words
The method of claim 56 wherein said chain-terminating nucleoside triphosphate is a labeled dideoxynucleoside triphosphate and wherein said step of identifying includes identifying said one or more nucleotides by the label on the labeled dideoxynucleoside triphosphate incorporated into said 3' recessed strand of said polynucleotide.
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| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
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