US5679522A — Methods of analysis and manipulation of DNA utili…

Claim 1

Independent Claim 149 words

A method for detecting a base pair mismatch in a DNA duplex, comprising the steps of: contacting at least one strand of a first DNA molecule with the complimentary strand of a second DNA molecule under conditions such that base pairing occurs; contacting a DNA duplex potentially containing a base pair mismatch with a mispair recognition protein under conditions suitable for said protein to form a specific complex only with said DNA duplex having a base pair mismatch, and not with a DNA duplex with conventional Watson-Crick base pairing, detecting any DNA:protein complexes as a measure of the presence of a base pair mismatch in said DNA duplex by contacting said DNA:protein complexes with a selectively adsorbent agent under conditions such that said DNA:protein complexes are retained on said agent while DNA not complexed with protein is not retained, and measuring the amount of DNA in said retained complexes.

Claim 2

Independent Claim 149 words

A method for detecting a base pair mismatch in a DNA duplex, comprising the steps of: contacting at least one strand of a first DNA molecule with the complimentary strand of a second DNA molecule under conditions such that base pairing occurs; contacting a DNA duplex potentially containing a base pair mismatch with a mispair recognition protein under conditions suitable for said protein to form a specific complex only with said DNA duplex having a base pair mismatch, and not with a DNA duplex with conventional Watson-Crick base pairing, detecting any DNA:protein complexes as a measure of the presence of a base pair mismatch in said DNA duplex by contacting said DNA:protein complexes with a selectively adsorbent agent under conditions such that said DNA:protein complexes are retained on said agent while DNA not complexed with protein is not retained, and measuring the amount of DNA in said retained complexes.

Claim 3

Dependent Claim 14 words

The method of claim 1 wherein said adsorbent agent is a membranous nitrocellulose filter.

Claim 4

Independent Claim 122 words

A method for detecting a base pair mismatch in a DNA duplex, comprising the steps of: contacting at least one strand of a first DNA molecule with the complimentary strand of a second DNA molecule under conditions such that base pairing occurs; contacting a DNA duplex potentially containing a base pair mismatch with a mispair recognition protein under conditions suitable for said protein to form a specific complex only with said DNA duplex having a base pair mismatch, and not with a DNA duplex with conventional Watson-Crick base pairing, detecting any DNA:protein complexes as a measure of the presence of a base pair mismatch in said DNA duplex by comparing the electrophoretic mobility of said DNA:protein complexes to that of uncomplexed DNA.

Claim 5

Independent Claim 122 words

A method for detecting a base pair mismatch in a DNA duplex, comprising the steps of: contacting at least one strand of a first DNA molecule with the complimentary strand of a second DNA molecule under conditions such that base pairing occurs; contacting a DNA duplex potentially containing a base pair mismatch with a mispair recognition protein under conditions suitable for said protein to form a specific complex only with said DNA duplex having a base pair mismatch, and not with a DNA duplex with conventional Watson-Crick base pairing, detecting any DNA:protein complexes as a measure of the presence of a base pair mismatch in said DNA duplex by comparing the electrophoretic mobility of said DNA:protein complexes to that of uncomplexed DNA.

Claim 6

Independent Claim 137 words

A method for detecting a base pair mismatch in a DNA duplex, comprising the steps of: contacting at least one strand of a first DNA molecule with the complimentary strand of a second DNA molecule under conditions such that base pairing occurs; contacting a DNA duplex potentially containing a base pair mismatch with a mispair recognition protein wherein said mispair recognition protein is the product of the mutS gene of Salmonella typhimurium, the hexA gene of Salmonella pneumoniae or the MsH1; MsH2, genes of yeast, under conditions suitable for said protein to form a specific complex only with said DNA duplex having a base pair mismatch and not with a DNA duplex with conventional Watson-Crick base pairing, and detecting any said complex as a measure of the presence of a base pair mismatch in said DNA duplex.

Claim 7

Independent Claim 137 words

A method for detecting a base pair mismatch in a DNA duplex, comprising the steps of: contacting at least one strand of a first DNA molecule with the complimentary strand of a second DNA molecule under conditions such that base pairing occurs; contacting a DNA duplex potentially containing a base pair mismatch with a mispair recognition protein wherein said mispair recognition protein is the product of the mutS gene of Salmonella typhimurium, the hexA gene of Salmonella pneumoniae or the MsH1; MsH2, genes of yeast, under conditions suitable for said protein to form a specific complex only with said DNA duplex having a base pair mismatch and not with a DNA duplex with conventional Watson-Crick base pairing, and detecting any said complex as a measure of the presence of a base pair mismatch in said DNA duplex.

Claim 8

Independent Claim 119 words

A method for detecting a base pair mismatch in a DNA duplex, comprising the steps of: contacting at least one strand of a first DNA molecule with the complimentary strand of a second DNA molecule under conditions such that base pairing occurs; contacting a DNA duplex potentially containing a base pair mismatch with a mispair recognition protein under conditions suitable for said protein to form a specific complex only with said DNA duplex having a base pair mismatch, and not with a DNA duplex with conventional Watson-Crick base pairing, detecting any DNA:protein complexes as a measure of the presence of a base pair mismatch in said DNA duplex by utilizing an antibody specific for said base mispair recognition protein.

Claim 9

Independent Claim 119 words

A method for detecting a base pair mismatch in a DNA duplex, comprising the steps of: contacting at least one strand of a first DNA molecule with the complimentary strand of a second DNA molecule under conditions such that base pairing occurs; contacting a DNA duplex potentially containing a base pair mismatch with a mispair recognition protein under conditions suitable for said protein to form a specific complex only with said DNA duplex having a base pair mismatch, and not with a DNA duplex with conventional Watson-Crick base pairing, detecting any DNA:protein complexes as a measure of the presence of a base pair mismatch in said DNA duplex by utilizing an antibody specific for said base mispair recognition protein.

Claim 10

Independent Claim 92 words

A method for detecting and localizing a base pair mismatch in a DNA duplex, comprising the steps of: contacting at least one strand of the first DNA molecule with the complementary strand of the second DNA molecule under conditions such that base pairing occurs; contacting the resulting double-stranded DNA duplexes with a mispair recognition protein under conditions such that the protein forms specific complexes with mispairs, subjecting said duplex molecules to hydrolysis with an exonuclease under conditions such that said complex blocks hydrolysis, and determining the location of said block to hydrolysis.

Claim 11

Independent Claim 92 words

A method for detecting and localizing a base pair mismatch in a DNA duplex, comprising the steps of: contacting at least one strand of the first DNA molecule with the complementary strand of the second DNA molecule under conditions such that base pairing occurs; contacting the resulting double-stranded DNA duplexes with a mispair recognition protein under conditions such that the protein forms specific complexes with mispairs, subjecting said duplex molecules to hydrolysis with an exonuclease under conditions such that said complex blocks hydrolysis, and determining the location of said block to hydrolysis.

Claim 12

Independent Claim 99 words

A method for detecting and localizing a base pair mismatch in a DNA duplex, comprising the steps of contacting at least one strand of a first DNA molecule with the complementary strand of a second DNA molecule under conditions such that base pairing occurs; contacting the resulting double-stranded DNA duplexes with a mispair recognition protein under conditions such that the protein forms specific complexes with mispairs and thereby directs modification of at least one strand of the DNA in the resulting DNA:protein complexes in the vicinity of the DNA:protein complex, and determining the location of the resulting DNA modification.

Claim 13

Independent Claim 99 words

A method for detecting and localizing a base pair mismatch in a DNA duplex, comprising the steps of contacting at least one strand of a first DNA molecule with the complementary strand of a second DNA molecule under conditions such that base pairing occurs; contacting the resulting double-stranded DNA duplexes with a mispair recognition protein under conditions such that the protein forms specific complexes with mispairs and thereby directs modification of at least one strand of the DNA in the resulting DNA:protein complexes in the vicinity of the DNA:protein complex, and determining the location of the resulting DNA modification.

Claim 14

Dependent Claim 28 words

The method of claim 7, wherein said mispair recognition protein is the product of the mutS gene of Escherichia coli, an homologous protein, or a functionally equivalent protein.

Claim 15

Dependent Claim 94 words

The method of claim 7, wherein the step for modifying the DNA duplex in the vicinity of the complexed protein comprises contacting said complexes with a defined mismatch correction system or subset comprising the following purified components: Escherichia coli MutH, and MutL, proteins, or species variations thereof, DNA helicase II, single-stranded DNA binding protein, DNA polymerase III holoenzyme, exonuclease I, exonuclease VII or RecJ exonuclease, ATP and one or more dideoxynucleoside triphosphates, under conditions that produce a single-stranded gap in one or both strands of the DNA duplex in the vicinity of the mismatch.

Claim 16

Dependent Claim 46 words

The method of claim 9 wherein said step for determining the location of said single-stranded gaps within said DNA duplex further includes the steps of cleaving said DNA with a single-stranded specific endonuclease, and comparing the electrophoretic mobilities of said cleaved fragments with unmodified DNA fragments.

Claim 17

Dependent Claim 52 words

The method of claim 7, wherein the step for modifying the DNA duplex in the vicinity of the complexed protein comprises: contacting said complexes with proteins of a mismatch repair system, ATP and a divalent cation under conditions such that an endonucleolytic incision is introduced in one strand of the duplex molecule.

Claim 18

Dependent Claim 26 words

The method of claim 11, wherein the step of determining the location comprises size fractionation by electrophoretic mobility under denaturing condition relative to unmodified DNA fragments.

Claim 19

Dependent Claim 28 words

The method of claim 11, wherein said proteins of a mismatch repair system are the MutH, MutL, and MutS proteins of the Escherichia coli methyl-directed mismatch repair system.

Claim 20

Independent Claim 106 words

A method for detecting and localizing a base pair mismatch within a DNA duplex, comprising the steps of: contacting at least one strand of a first DNA molecule with a complementary strand of a second DNA molecule under conditions such that base pairing occurs; contacting resultant duplex DNA molecules with a mispair recognition protein able to cleave DNA, under conditions such that the protein forms specific complexes with a mispair and thereby directs cleavage of at least one strand of the DNA in the resulting DNA protein complexes in the vicinity of the DNA protein complex, and determining the location of the cleavage of the DNA.

Claim 21

Independent Claim 106 words

A method for detecting and localizing a base pair mismatch within a DNA duplex, comprising the steps of: contacting at least one strand of a first DNA molecule with a complementary strand of a second DNA molecule under conditions such that base pairing occurs; contacting resultant duplex DNA molecules with a mispair recognition protein able to cleave DNA, under conditions such that the protein forms specific complexes with a mispair and thereby directs cleavage of at least one strand of the DNA in the resulting DNA protein complexes in the vicinity of the DNA protein complex, and determining the location of the cleavage of the DNA.

Claim 22

Independent Claim 88 words

A method for detecting and localizing A--G mispairs in a DNA duplex, comprising the steps of: contacting at least one strand of the first DNA molecule with the complementary strand of the second DNA molecule under conditions such that base pairing occurs; contacting resultant duplex DNA molecules with a mispair recognition protein that recognizes A--G mispairs and an apurinic endonuclease or lyase under conditions such that in the presence of a mismatch an endonucleolytic incision is introduced in the duplex molecule, and determining the location of the incision.

Claim 23

Independent Claim 88 words

A method for detecting and localizing A--G mispairs in a DNA duplex, comprising the steps of: contacting at least one strand of the first DNA molecule with the complementary strand of the second DNA molecule under conditions such that base pairing occurs; contacting resultant duplex DNA molecules with a mispair recognition protein that recognizes A--G mispairs and an apurinic endonuclease or lyase under conditions such that in the presence of a mismatch an endonucleolytic incision is introduced in the duplex molecule, and determining the location of the incision.

Claim 24

Dependent Claim 21 words

The method of claim 15, wherein said A--G mispair recognition protein is the product of the mutY gene of Escherichia coli.

Claim 25

Dependent Claim 12 words

The method of claim 15, wherein the analytical method comprises gel electrophoresis.

Claim 26

Independent Claim 81 words

The method of claims 6, 7, 14, or 15, wherein the DNA molecules are obtained from the following sources: different individuals of the same species, individuals of different species, individuals of different kingdoms, different tissue types, the same tissue type in different states of growth, different cell types, cells of the same type in different states of growth, cells of the same origin in different stages of development and cells of the same type that may have undergone differential somatic mutagenesis.

Claim 27

Dependent Claim 28 words

The method of claim 18, wherein one of the DNA molecules comprises a probe sequence that has been at least partially characterized by restriction mapping or sequence analysis.

Claim 28

Dependent Claim 38 words

Assay kit for detecting and localizing individual base sequence differences within homologous regions of two DNA molecules comprising: an aliquot of a mispair recognition protein attached to a hydroxyl radical cleaving function or attached to a DNA endonuclease.

Claim 29

Dependent Claim 38 words

Assay kit for detecting and localizing individual base sequence differences within homologous regions of two DNA molecules comprising: an aliquot of a mispair recognition protein attached to a hydroxyl radical cleaving function or attached to a DNA endonuclease.

Claim 30

Dependent Claim 20 words

The kit of claim 20, wherein the mispair recognition protein is the product of the mutS gene of Escherichia coli.

Claim 31

Dependent Claim 38 words

Assay kit for detecting and localizing an A--G mispair within a DNA duplex comprising one or more of the following components; an aliquot of an A--G mispair recognition protein; and an aliquot of an apurinic endonuclease or lyase.

Claim 32

Dependent Claim 38 words

Assay kit for detecting and localizing an A--G mispair within a DNA duplex comprising one or more of the following components; an aliquot of an A--G mispair recognition protein; and an aliquot of an apurinic endonuclease or lyase.

Claim 33

Dependent Claim 21 words

The kit of claim 22, wherein the A--G mispair recognition protein is the product of the MutY gene of Escherichia coli.

Claim 34

Dependent Claim 20 words

The kit of claim 20, wherein the mispair recognition protein is the product of the mutS gene of Escherichia coli.

Claim 35

Dependent Claim 12 words

The method of claim 9, wherein said exogenous dideoxynucleoside triphosphates are omitted.

Annuity Stage	Window Opens	Payment Deadline	Surcharge Period Until	Estimated Fee	Status
3.5 Years (Year 4)	2000-10-20	2001-04-21	2001-10-21	$2000.00	Window Closed / Lapsed
7.5 Years (Year 8)	2004-10-20	2005-04-21	2005-10-21	$3760.00	Window Closed / Lapsed
11.5 Years (Year 12)	2008-10-20	2009-04-21	2009-10-21	$7700.00	Window Closed / Lapsed

Methods of analysis and manipulation of DNA utilizing mismatch repair systems

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