US4794075A
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Method for locating and purifying DNA containing single base mismatches
Assignee: LIFECODES Inc A CORP OF NEW YORK
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Filing: 1985-08-29
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Granted: 1988-12-27
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Expires: 2005-08-29
Abstract
Abstract A method for distinguishing fragments of DNA which contain single base mismatches from their perfectly paired homologues is disclosed. Single stranded regions within a duplex fragment are modified with carbodiimide, which reacts with unpaired guanine (G) and thymine (T) residues in DNA. Linear duplex DNA molecules do not react, while DNA molecules with single base mismatches react quantitatively with carbodiimide. Following reaction with carbodiimide, the DNA molecules are fractionated on high percentage polyacrylamide gels such that modified and unmodified fragments can be clearly distinguished. Application of this technique in order to located and purify DNA sequence differences responsible for phenotype variation and inherited disease is disclosed.
Inventors
- 👤 John P. Ford
- 👤 David F. Novack
- 👤 Nancy J. Casna
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-30 07:40
CPC Classifications
G; G01; G01N; G01N27/00; G01N27/26; G01N27/416; G01N27/447; C; C12; C12Q; C12Q1/00; C12Q1/68; C12Q1/6813; C12Q1/6827; Y; Y10; Y10S; Y10S435/00; Y10S435/803
Claims Analysis
Total Claims: 44
Claim 1
Independent Claim
171 words
A method for detecting guanine and thymine bases which are unpaired according to the Watson-Crick base pairing scheme in a double stranded polynucleotide molecule, each unpaired guanine or thymine base being immediately preceded by at least one base which is paired, and immediately followed by at least one base which is paired, said preceding and following paired bases being on the same polynucleotide sequence as the unpaired guanine or thymine base comprising: (a) reacting the double stranded polynucleotide molecule with a reagent capable of altering the electrophoretic mobility of a double stranded polynucleotide molecule by derivatizing unpaired guanine and thymine bases in the double stranded polynucleotide molecule, wherein the double strandned polynucleotide molecule is not a covalently closed circular DNA; (b) observing the electrophoretic mobility of the double stranded polynucleotide molecule which has been reacted with the reagent; and (c) determining the presence or absence of an alteration in the electrophoretic mobility; whereby the presence or absence of unpaired guanine and thymine bases in the double stranded polynucleotide molecule is detected.
Claim 2
Independent Claim
171 words
A method for detecting guanine and thymine bases which are unpaired according to the Watson-Crick base pairing scheme in a double stranded polynucleotide molecule, each unpaired guanine or thymine base being immediately preceded by at least one base which is paired, and immediately followed by at least one base which is paired, said preceding and following paired bases being on the same polynucleotide sequence as the unpaired guanine or thymine base comprising: (a) reacting the double stranded polynucleotide molecule with a reagent capable of altering the electrophoretic mobility of a double stranded polynucleotide molecule by derivatizing unpaired guanine and thymine bases in the double stranded polynucleotide molecule, wherein the double strandned polynucleotide molecule is not a covalently closed circular DNA; (b) observing the electrophoretic mobility of the double stranded polynucleotide molecule which has been reacted with the reagent; and (c) determining the presence or absence of an alteration in the electrophoretic mobility; whereby the presence or absence of unpaired guanine and thymine bases in the double stranded polynucleotide molecule is detected.
Claim 3
Independent Claim
183 words
A method for detecting guanine and thymine bases which are unpaired according to the Watson-Crick base pairing scheme in a double stranded polynucleotide molecule, wherein each unpaired guanine or thymine base being immediately preceded by at least one base which is paired and immediately followed by at least one base which is paired, said preceding and following paired bases being on the same polynucleotide sequence as the unpaired guanine or thymine base comprising: (a) hybridizing a first single stranded polynucleotide sequence with a second single stranded polynucleotide sequence to form a double stranded polynucleotide molecule; (b) reacting the double stranded polynucleotide molecule with a reagent capable of altering the electrophoretic mobility of a double stranded polynucleotide molecule by derivatizing unpaired guanine and thymine bases in the double stranded polynucleotide molecule; (c) observing the state of electrophoretic mobility the double stranded polynucleotide molecule which has been reacted with the reagent; (d) determining the presence or absence of an alteration in the state of the property, wherein the presence or absence of unpaired guanine and thymine bases in the double stranded polynucletide molecule is detected.
Claim 4
Independent Claim
183 words
A method for detecting guanine and thymine bases which are unpaired according to the Watson-Crick base pairing scheme in a double stranded polynucleotide molecule, wherein each unpaired guanine or thymine base being immediately preceded by at least one base which is paired and immediately followed by at least one base which is paired, said preceding and following paired bases being on the same polynucleotide sequence as the unpaired guanine or thymine base comprising: (a) hybridizing a first single stranded polynucleotide sequence with a second single stranded polynucleotide sequence to form a double stranded polynucleotide molecule; (b) reacting the double stranded polynucleotide molecule with a reagent capable of altering the electrophoretic mobility of a double stranded polynucleotide molecule by derivatizing unpaired guanine and thymine bases in the double stranded polynucleotide molecule; (c) observing the state of electrophoretic mobility the double stranded polynucleotide molecule which has been reacted with the reagent; (d) determining the presence or absence of an alteration in the state of the property, wherein the presence or absence of unpaired guanine and thymine bases in the double stranded polynucletide molecule is detected.
Claim 5
Dependent Claim
21 words
The method of claim 2 wherein the unpaired guanine and thymine bases are mispaired according to the Watson-Crick base pairing scheme.
Claim 6
Dependent Claim
61 words
The method of claim 2 wherein each unpaired guanine or thymine base is immediately preceded by a first plurality of bases all of which are paired and immediately followed by a second plurality of bases all of which are paired, said preceding and following pluralities of paired bases being on the same polynucleotide sequence as the unpaired guanine or thymine base.
Claim 7
Dependent Claim
21 words
The method of claim 4 wherein the first plurality is at least 40 and the second plurality is at least 40.
Claim 8
Dependent Claim
11 words
The method of claim 2 wherein the reagent is a carbodiimide.
Claim 9
Dependent Claim
50 words
The method of claim 6 wherein: (i) step (b) comprises reacting the double stranded polynucleotide molecule with about 0.1M carbodiimide at about pH 8.5, for about 4 hours at a temperature between about 25° C. and about 40° C.; and (ii) the first sequence and the second sequence are DNA.
Claim 10
Dependent Claim
11 words
The method of claim 7 wherein the carbodiimide is 1-cyclohexyl-3-(2-morpholino-ethyl) carbodiimide.
Claim 11
Dependent Claim
15 words
The method of claim 2 wherein the first sequence and the second sequence are DNA.
Claim 12
Independent Claim
151 words
A method for purifying double stranded polynucleotide molecules that are base paired throughout the entire molecules according to the Watson-Crick scheme comprising: (a) hybridizing a mixture of molecules comprising single stranded polynucleotide sequences to form double stranded polynucleotide molecules; (b) reacting the double stranded polynucleotide molecules with a reagent capable of altering the electrophoretic mobility of a double stranded polynucleotide molecule by derivatizing unpaired guanine and thymine bases in the double stranded polynucleotide molecule, each unpaired guanine or thymine base being immediately preceded by at least one base which is paired and immediately followed by at least one base which is paired, said preceding and following paired bases being on the same polynucleotide sequence as the unpaired guanine or thymine base; (c) selectively separating underivatized polynucleotide molecules from derivatized polynucleotide molecules according to the electrophoretic mobility, whereby double stranded polynucleotide molecules that are based paired throughout the entire molecules are purified.
Claim 13
Independent Claim
151 words
A method for purifying double stranded polynucleotide molecules that are base paired throughout the entire molecules according to the Watson-Crick scheme comprising: (a) hybridizing a mixture of molecules comprising single stranded polynucleotide sequences to form double stranded polynucleotide molecules; (b) reacting the double stranded polynucleotide molecules with a reagent capable of altering the electrophoretic mobility of a double stranded polynucleotide molecule by derivatizing unpaired guanine and thymine bases in the double stranded polynucleotide molecule, each unpaired guanine or thymine base being immediately preceded by at least one base which is paired and immediately followed by at least one base which is paired, said preceding and following paired bases being on the same polynucleotide sequence as the unpaired guanine or thymine base; (c) selectively separating underivatized polynucleotide molecules from derivatized polynucleotide molecules according to the electrophoretic mobility, whereby double stranded polynucleotide molecules that are based paired throughout the entire molecules are purified.
Claim 14
Dependent Claim
21 words
The method of claim 10 wherein the unpaired guanine and thymine bases are mispaired according to the Watson-Crick base pairing scheme.
Claim 15
Dependent Claim
60 words
The method of claim 10 wherein each unpaired guanine or thymine base is immediately preceded by a plurality of bases all of which are paired and immediately followed by a second plurality of bases all of which are paired, said preceding and following pluralities of paired bases being on the same polynucleotide sequence as the unpaired guanine or thymine base.
Claim 16
Dependent Claim
21 words
The method of claim 12 wherein the first plurality is at least 40 and the second plurality is at least 40.
Claim 17
Dependent Claim
27 words
The method of claim 10 wherein step (b) comprises reacting the double stranded polynucleotide molecules with about 0.1M carbodiimide at about pH 8.5, for about 4 hours.
Claim 18
Dependent Claim
28 words
The method of claim 10 wherein step (c) comprises molecularly cloning and biologically selecting the underivatized polynucleotide molecules in an environment which is incapable of replicating derivatized DNA.
Claim 19
Dependent Claim
12 words
The method of claim 15 wherein the derivatized DNA is carbodiimide-derivatized DNA.
Claim 20
Dependent Claim
30 words
The method of claim 16 wherein the environment incapable of replicating derivatized DNA is the cellular environment of replicating derivatized DNA is the cellular environment of a UVRA, RecA host.
Claim 21
Dependent Claim
107 words
The method of claim 10 wherein: (A) step (b) comprises reacting the double stranded polynucleotide molecules with about 0.1M carbodiimide at about pH 8.5, for about 4 hours; (B) step (c) comprises: (i) electrophoresing the double stranded polynucleotide molecules which have been reacted with carbodiimide in a first dimension; (ii) removing the carbodiimide which has become bound to double stranded polynucleotide molecules by treating the double stranded polynucleotides with alkali; (iii) further electrophoresing the double stranded polynucleotide molecules in a second dimension; and (iv) identifying and obtaining the double stranded polynucleotide molecules which have electrophoretic mobilities in step (iii) different from their electrophoretic mobilities in step (i).
Claim 22
Dependent Claim
25 words
The method according to claim 10 wherein the mixture of molecules comprising single stranded polynucleotide sequences is obtained from two individuals having a common ancestor.
Claim 23
Dependent Claim
16 words
The method of claim 19 wherein the number of generations separating the two individuals is six.
Claim 24
Dependent Claim
22 words
The method of claim 19 wherein the two individuals and their common ancestors are predisposed to, have or had a genetic disease.
Claim 25
Dependent Claim
23 words
The method of claim 19 wherein the genetic disease is selected from the group consisting of breast cancer, colon cancer, and cystic fibrosis.
Claim 26
Independent Claim
167 words
A method for detecting the presence or absence of bases which are mispaired according to the Watson-Crick base pairing scheme in a double stranded polynucleotide molecule, and which mispaired bases are immediately preceded by at least one base which is paired, and immediately followed by at least one base which is paired, said preceding and following paired bases being on the same polynucleotide sequence as the unpaired base, comprising: (a) reacting the double stranded polynucleotide molecule with a reagent capable of altering the electrophoretic mobility of a double stranded polynucleotide molecule by derivatizing at least one mispaired baes in the double stranded polynucleotide molecule, wherein the double stranded polynucleotide molecule is not a covalently closed circular DNA; (b) observing the electrophoretic mobility of the double stranded polynucleotide molecule which has been reacted with the reagent; (c) determining the presence or absence of an alteration in the electrophoretic mobility, whereby the presence or absence of at least one mispaired base in the double stranded polynucleotide molecule is detected.
Claim 27
Independent Claim
167 words
A method for detecting the presence or absence of bases which are mispaired according to the Watson-Crick base pairing scheme in a double stranded polynucleotide molecule, and which mispaired bases are immediately preceded by at least one base which is paired, and immediately followed by at least one base which is paired, said preceding and following paired bases being on the same polynucleotide sequence as the unpaired base, comprising: (a) reacting the double stranded polynucleotide molecule with a reagent capable of altering the electrophoretic mobility of a double stranded polynucleotide molecule by derivatizing at least one mispaired baes in the double stranded polynucleotide molecule, wherein the double stranded polynucleotide molecule is not a covalently closed circular DNA; (b) observing the electrophoretic mobility of the double stranded polynucleotide molecule which has been reacted with the reagent; (c) determining the presence or absence of an alteration in the electrophoretic mobility, whereby the presence or absence of at least one mispaired base in the double stranded polynucleotide molecule is detected.
Claim 28
Independent Claim
176 words
A method for detecting the presence or absence of bases which are mispaired according to the Watson-Crick base pairing scheme in a double stranded polynucleotide molecule, and which mispaired bases are immediately preceded by at least one base which is paired, and immediately followed by at least one base which is paired, said preceding and following paired bases being on the same polynucleotide sequence as the unpaired base, comprising: (a) hybridizing a first single stranded polynucleotide sequence with a second single stranded poyynucleotide sequence to form a double stranded polynucleotide molecule; (b) reacting the double stranded polynucleotide molecule with a reagent capable of altering the electrophoretic mobility of a double stranded polynucleotide molecule by derivatizing at least one mispaired base in the double stranded polynucleotide molecule; (c) observing the electrophoretic mobility of the double stranded polynucleotide molecule which has been reacted with the reagent; (d) determining the presence or absence of an alteration in the electrophoretic mobility, whereby the presence or absence of at least one mispaired base in the double stranded polynucleotide molecule is detected.
Claim 29
Independent Claim
176 words
A method for detecting the presence or absence of bases which are mispaired according to the Watson-Crick base pairing scheme in a double stranded polynucleotide molecule, and which mispaired bases are immediately preceded by at least one base which is paired, and immediately followed by at least one base which is paired, said preceding and following paired bases being on the same polynucleotide sequence as the unpaired base, comprising: (a) hybridizing a first single stranded polynucleotide sequence with a second single stranded poyynucleotide sequence to form a double stranded polynucleotide molecule; (b) reacting the double stranded polynucleotide molecule with a reagent capable of altering the electrophoretic mobility of a double stranded polynucleotide molecule by derivatizing at least one mispaired base in the double stranded polynucleotide molecule; (c) observing the electrophoretic mobility of the double stranded polynucleotide molecule which has been reacted with the reagent; (d) determining the presence or absence of an alteration in the electrophoretic mobility, whereby the presence or absence of at least one mispaired base in the double stranded polynucleotide molecule is detected.
Claim 30
Dependent Claim
11 words
The method of claim 24 wherein the reagent is a carbodiimide.
Claim 31
Dependent Claim
50 words
The method of claim 25 wherein: (i) step (b) comprises reacting the double stranded polynucleotide molecule with about 0.1M carbodiimide at about pH 8.5, for about 4 hours at a temperature between about 25° C. and about 40° C.; and (ii) the first sequence and the second sequence are DNA.
Claim 32
Independent Claim
146 words
A method for purifying double stranded polynucleotide molecules that are base paired throughout the entire molecules according to the Watson-Crick scheme comprising: (a) hybridizing a mixture of molecules comprising single stranded polynucleotide sequences to form double stranded polynucleotide molecules; (b) reacting the double standed polynucleotide molecules with a reagent capable of altering the elelctrophoretic mobility of a double stranded polynucleotide molecule by derivatizing at least one mispaired base in the double stranded polynucleotide molecule, wherein each mispaired base is immediately preceded by at least one base which is paired and immediately followed by at least one base which is paired, said preceding and following paired bases being on the same polynucleotide sequence as said mispaired base; (c) selectively separating underivatized polynucleotide molecules from derivatized polynucleotide molecules according to the electrophoretic mobility, whereby double stranded polynucleotide molecules that are based paired throughout the entire molecules are purified.
Claim 33
Independent Claim
146 words
A method for purifying double stranded polynucleotide molecules that are base paired throughout the entire molecules according to the Watson-Crick scheme comprising: (a) hybridizing a mixture of molecules comprising single stranded polynucleotide sequences to form double stranded polynucleotide molecules; (b) reacting the double standed polynucleotide molecules with a reagent capable of altering the elelctrophoretic mobility of a double stranded polynucleotide molecule by derivatizing at least one mispaired base in the double stranded polynucleotide molecule, wherein each mispaired base is immediately preceded by at least one base which is paired and immediately followed by at least one base which is paired, said preceding and following paired bases being on the same polynucleotide sequence as said mispaired base; (c) selectively separating underivatized polynucleotide molecules from derivatized polynucleotide molecules according to the electrophoretic mobility, whereby double stranded polynucleotide molecules that are based paired throughout the entire molecules are purified.
Claim 34
Dependent Claim
55 words
The method of claim 27 wherein each mispaired base is immediately preceded by a first plurality of bases all of which are paired and immediately followed by a second plurality of bases all of which are paired, said preceding and following pluralities of paired bases being on the same polynucleotide sequence as said mispaired base.
Claim 35
Dependent Claim
21 words
The method of claim 28 wherein the first plurality is at least 40 and the second plurality is at least 40.
Claim 36
Dependent Claim
27 words
The method of claim 27 wherein step (b) comprises reacting the double stranded polynucleotide molecules with about 0.1M carbodiimide at about pH 8.5, for about 4 hours.
Claim 37
Dependent Claim
28 words
The method of claim 27 wherein step (c) comprises molecularly cloning and biologically selecting the underivatized polynucleotide molecules in an environment which is incapable of replicating derivatized DNA.
Claim 38
Dependent Claim
12 words
The method of claim 31 wherein the derivatized DNA is carbodiimide-derivatized DNA.
Claim 39
Dependent Claim
22 words
The method of claim 32 wherein the environment incapable of replicating derivatized DNA is the cellular environment of a UVRA, RecA host.
Claim 40
Dependent Claim
107 words
The method of claim 27 wherein: (A) step (b) comprises reacting the double stranded polynucleotide molecules with about 0.1M carbodiimide at about pH 8.5, for about 4 hours; (B) step (c) comprises: (i) electrophoresing the double stranded polynucleotide molecules which have been reacted with carbodiimide in a first dimension; (ii) removing the carbodiimide which has become bound to double stranded polynucleotide molecules by treating the double stranded polynucleotides with alkali; (iii) further electrophoresing the double stranded polynucleotide molecules in a second dimension; and (iv) identifying and obtaining the double stranded polynucleotide molecules which have electrophoretic mobilities in step (iii) different from their electrophoretic mobilities in step (i).
Claim 41
Dependent Claim
25 words
The method according to claim 27 wherein the mixture of molecules comprising single stranded polynucleotide sequences is obtained from two individuals having a common ancestor.
Claim 42
Dependent Claim
16 words
The method of claim 35 wherein the number of generations separating the two individuals is six.
Claim 43
Dependent Claim
22 words
The method of claim 35 wherein the two individuals and their common ancestors are predisposed to, have or had a genetic disease.
Claim 44
Dependent Claim
23 words
The method of claim 35 wherein the genetic disease is selected from the group consisting of breast cancer, colon cancer, and cystic fibrosis.
References Cited (Backward Citations)
No citation records indexed.
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| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
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| 3.5 Years (Year 4) | 1991-12-27 | 1992-06-27 | 1992-12-27 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1995-12-27 | 1996-06-27 | 1996-12-27 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1999-12-27 | 2000-06-27 | 2000-12-27 | $7700.00 | Window Closed / Lapsed |