US5556750A — Methods and kits for fractionating a population o…

Claim 1

Independent Claim 150 words

A method for eliminating DNA molecules containing one or more base pairing mismatches from a population of heterohybrid duplex DNA molecules formed by base pairing of single-stranded DNA molecules obtained from a first source and a second source, comprising the steps of: digesting genomic DNA from said first and said second source with a restriction endonuclease, methylating the DNA from one of said sources, denaturing said DNA from said first and said second source, mixing DNA from said first and said second source in the presence of a recombinase protein, proteins of a mismatch repair system that modulate said recombinase protein, single-strand binding protein, and ATP, such that DNA duplexes form in homologous regions of DNA molecules from said first and said second source and the presence of a base pair mismatch results in regions that remain single-stranded, and removing molecules that contain a said single-stranded region from said population.

Claim 2

Independent Claim 150 words

A method for eliminating DNA molecules containing one or more base pairing mismatches from a population of heterohybrid duplex DNA molecules formed by base pairing of single-stranded DNA molecules obtained from a first source and a second source, comprising the steps of: digesting genomic DNA from said first and said second source with a restriction endonuclease, methylating the DNA from one of said sources, denaturing said DNA from said first and said second source, mixing DNA from said first and said second source in the presence of a recombinase protein, proteins of a mismatch repair system that modulate said recombinase protein, single-strand binding protein, and ATP, such that DNA duplexes form in homologous regions of DNA molecules from said first and said second source and the presence of a base pair mismatch results in regions that remain single-stranded, and removing molecules that contain a said single-stranded region from said population.

Claim 3

Dependent Claim 15 words

The method of claim 1, wherein said recombinase protein is the Escherichia coli RecA protein.

Claim 4

Dependent Claim 26 words

The method of claim 1, wherein said mismatch repair system comprises the Escherichia coli methyl-directed mismatch repair system and consists of the MutS and MutL proteins.

Claim 5

Independent Claim 161 words

A method for eliminating DNA molecules containing one or more mismatches from a population of heterohybrid duplex DNA molecules formed by a strand transfer reaction between duplex DNA molecules obtained from a first source and denatured DNA molecules obtained from a second source, comprising the steps of: restriction digesting genomic DNA from said first and said second sources, methylating the DNA of said first or said second source, denaturing DNA from said second source, mixing DNA molecules from said first and said second source in the presence of a protein which catalyzes a strand transfer reaction, proteins of a mismatch repair system that modulate said protein which catalyzes a strand transfer reaction, single-strand binding protein, and ATP, such that DNA heteroduplexes form in homologous regions of DNA molecules from the first and the second source and the presence of a base pair mismatch results in regions that remain single-stranded, and removing molecules that contain a said single-stranded region from said population.

Claim 6

Independent Claim 161 words

A method for eliminating DNA molecules containing one or more mismatches from a population of heterohybrid duplex DNA molecules formed by a strand transfer reaction between duplex DNA molecules obtained from a first source and denatured DNA molecules obtained from a second source, comprising the steps of: restriction digesting genomic DNA from said first and said second sources, methylating the DNA of said first or said second source, denaturing DNA from said second source, mixing DNA molecules from said first and said second source in the presence of a protein which catalyzes a strand transfer reaction, proteins of a mismatch repair system that modulate said protein which catalyzes a strand transfer reaction, single-strand binding protein, and ATP, such that DNA heteroduplexes form in homologous regions of DNA molecules from the first and the second source and the presence of a base pair mismatch results in regions that remain single-stranded, and removing molecules that contain a said single-stranded region from said population.

Claim 7

Dependent Claim 25 words

The method of claim 1 or 4, wherein the removal of said molecules containing a single-stranded region is by treatment with a single-strand specific nuclease.

Claim 8

Dependent Claim 24 words

The method of claim 1 or 4, wherein the removal of said molecules containing a single-stranded region is by chromatography on benzoylated naphthoylated DEAE.

Claim 9

Dependent Claim 16 words

The method of claim 4, wherein said strand transferase protein is the Escherichia coli RecA protein.

Claim 10

Dependent Claim 25 words

The method of claim 4, wherein mismatch repair system comprises the Escherichia coli methyl-directed mismatch repair system and consists of the MutS and MutL proteins.

Claim 11

Dependent Claim 78 words

The method of claim 1 or 4, wherein said sources of DNA are selected from the group consisting of: 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 origin have undergone differential somatic mutagenesis.

Claim 12

Dependent Claim 22 words

The method of claim 9, wherein one of said sources consists of a probe sequence that has been at least partially characterized.

Claim 13

Independent Claim 88 words

A method of genomic mismatch scanning, wherein heterohybrid DNA molecules containing base pair mismatches are removed, without the use of exonuclease III, comprising the steps of: contacting a population of heterohybrid DNA molecules potentially containing a base pair mismatch with a DNA mismatch repair system in the presence of one or more dideoxynucleoside triphosphates such that a single-stranded region is generated in a DNA molecule that contained a base pair mismatch, without the use of exonuclease III, and, removing said molecule containing a single-stranded region from the population.

Claim 14

Independent Claim 88 words

A method of genomic mismatch scanning, wherein heterohybrid DNA molecules containing base pair mismatches are removed, without the use of exonuclease III, comprising the steps of: contacting a population of heterohybrid DNA molecules potentially containing a base pair mismatch with a DNA mismatch repair system in the presence of one or more dideoxynucleoside triphosphates such that a single-stranded region is generated in a DNA molecule that contained a base pair mismatch, without the use of exonuclease III, and, removing said molecule containing a single-stranded region from the population.

Claim 15

Dependent Claim 19 words

The method of claim 11, wherein said DNA mismatch repair system is the Escherichia coli methyl-directed mismatch repair system.

Claim 16

Dependent Claim 22 words

The method of claim 11, wherein the removal of said molecule containing a single-stranded region is by chromatography on benzoylated naphthoylated DEAE.

Claim 17

Dependent Claim 23 words

The method of claim 11, wherein the removal of said molecule containing a single-stranded region is by treatment with a single-strand specific nuclease.

Claim 18

Independent Claim 105 words

A method for fractionating a population of DNA molecules based upon a mismatch in a subset of said molecules, wherein a heterohybrid DNA molecule containing a base pair mismatch is separated from non-mismatch-containing molecules, without the use of exonuclease III, comprising the steps of: contacting a population of heterohybrid DNA molecules potentially containing a base pair mismatch with a DNA mismatch repair system and biotinylated nucleotide triphosphates, in the absence of exonuclease III, such that biotinylated nucleotides are incorporated into DNA molecules that contained a base pair mismatch and, separating said molecule containing biotinylated nucleotides from those not containing said nucleotides by binding to avidin.

Claim 19

Independent Claim 105 words

A method for fractionating a population of DNA molecules based upon a mismatch in a subset of said molecules, wherein a heterohybrid DNA molecule containing a base pair mismatch is separated from non-mismatch-containing molecules, without the use of exonuclease III, comprising the steps of: contacting a population of heterohybrid DNA molecules potentially containing a base pair mismatch with a DNA mismatch repair system and biotinylated nucleotide triphosphates, in the absence of exonuclease III, such that biotinylated nucleotides are incorporated into DNA molecules that contained a base pair mismatch and, separating said molecule containing biotinylated nucleotides from those not containing said nucleotides by binding to avidin.

Claim 20

Dependent Claim 19 words

The method of claim 15, wherein said DNA mismatch repair system is the Escherichia coli methyl-directed mismatch repair system.

Claim 21

Independent Claim 89 words

A method for removing DNA duplex molecules containing base pair mismatches in a population of heteroduplex DNA molecules produced from different sources, comprising the steps of: contacting said population of DNA duplex molecules potentially containing base pair mismatches with some or all components of a mismatch repair system and biotinylated nucleotides such that said nucleotides are incorporated into duplex molecules that contain a base pair mismatch and not into DNA duplex molecules lacking a base pair mismatch, and removing said duplexes with incorporated biotinylated nucleotides by binding to avidin.

Claim 22

Independent Claim 89 words

A method for removing DNA duplex molecules containing base pair mismatches in a population of heteroduplex DNA molecules produced from different sources, comprising the steps of: contacting said population of DNA duplex molecules potentially containing base pair mismatches with some or all components of a mismatch repair system and biotinylated nucleotides such that said nucleotides are incorporated into duplex molecules that contain a base pair mismatch and not into DNA duplex molecules lacking a base pair mismatch, and removing said duplexes with incorporated biotinylated nucleotides by binding to avidin.

Annuity Stage	Window Opens	Payment Deadline	Surcharge Period Until	Estimated Fee	Status
3.5 Years (Year 4)	1999-09-17	2000-03-18	2000-09-17	$2000.00	Window Closed / Lapsed
7.5 Years (Year 8)	2003-09-17	2004-03-18	2004-09-17	$3760.00	Window Closed / Lapsed
11.5 Years (Year 12)	2007-09-17	2008-03-18	2008-09-17	$7700.00	Window Closed / Lapsed

Methods and kits for fractionating a population of DNA molecules based on the presence or absence of a base-pair mismatch utilizing mismatch repair systems

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