US5552270A — Methods of DNA sequencing by hybridization based …

Claim 1

Independent Claim 157 words

A method for determining a DNA nucleotide sequence comprising: a) providing a solid support with an attached matrix; b) determining for each oligonucleotide member of a set of oligonucleotides to be bound to said matrix a concentration such that hybridization and washing of all fully complementary duplexes formed between said oligonucleotides and a labeled test DNA to be sequenced may be carried out at the same temperature, said concentration providing for the same dissociation temperature for all of said fully complementary duplexes; c) forming an array of said oligonucleotides within said matrix, each oligonucleotide added at said determined concentration; d) hybridizing said array of oligonucleotides with a labeled test DNA; e) washing in conditions ensuring that imperfect duplexes are dissociated; f) discriminating single-base substitutions in the test DNA by analyzing the distribution of the labeled test DNA; and g) determining the nucleotide sequence of the test DNA based on the results of the analysis in step f).

Claim 2

Independent Claim 157 words

A method for determining a DNA nucleotide sequence comprising: a) providing a solid support with an attached matrix; b) determining for each oligonucleotide member of a set of oligonucleotides to be bound to said matrix a concentration such that hybridization and washing of all fully complementary duplexes formed between said oligonucleotides and a labeled test DNA to be sequenced may be carried out at the same temperature, said concentration providing for the same dissociation temperature for all of said fully complementary duplexes; c) forming an array of said oligonucleotides within said matrix, each oligonucleotide added at said determined concentration; d) hybridizing said array of oligonucleotides with a labeled test DNA; e) washing in conditions ensuring that imperfect duplexes are dissociated; f) discriminating single-base substitutions in the test DNA by analyzing the distribution of the labeled test DNA; and g) determining the nucleotide sequence of the test DNA based on the results of the analysis in step f).

Claim 3

Dependent Claim 16 words

A method according to claim 1 wherein washing is carried out at a fixed temperature gradient.

Claim 4

Dependent Claim 32 words

A method according to claim 1 wherein dependence of an amount of remaining duplexes on temperature is determined during washing and compared with the dependence for a DNA of a known sequence.

Claim 5

Independent Claim 163 words

A method for determining a DNA nucleotide sequence comprising: a) providing a solid support with an attached matrix; b) using an array of oligonucleotides wherein for each oligonucleotide member of the array to be bound to said matrix, a concentration has been determined such that hybridization and washing of all fully complementary duplexes formed between said oligonucleotides and a labeled test DNA to be sequenced may be carried out at the same temperature, said concentration providing for the same dissociation temperature for all of said fully complementary duplexes; c) forming an array of said oligonucleotides within said matrix, each oligonucleotide added at said determined concentration; d) hybridizing said array of oligonucleotides with a labeled test DNA; e) washing in conditions ensuring that imperfect duplexes are dissociated; f) discriminating single-base substitutions in the test DNA by analyzing the distribution of the labeled test DNA; and g) determining the nucleotide sequence of the test DNA based on the results of the analysis in step f).

Claim 6

Independent Claim 163 words

A method for determining a DNA nucleotide sequence comprising: a) providing a solid support with an attached matrix; b) using an array of oligonucleotides wherein for each oligonucleotide member of the array to be bound to said matrix, a concentration has been determined such that hybridization and washing of all fully complementary duplexes formed between said oligonucleotides and a labeled test DNA to be sequenced may be carried out at the same temperature, said concentration providing for the same dissociation temperature for all of said fully complementary duplexes; c) forming an array of said oligonucleotides within said matrix, each oligonucleotide added at said determined concentration; d) hybridizing said array of oligonucleotides with a labeled test DNA; e) washing in conditions ensuring that imperfect duplexes are dissociated; f) discriminating single-base substitutions in the test DNA by analyzing the distribution of the labeled test DNA; and g) determining the nucleotide sequence of the test DNA based on the results of the analysis in step f).

Claim 7

Independent Claim 119 words

A device for determining a nucleotide sequence, said device comprising: (a) a solid support; and (b) a matrix affixed to said support, said matrix containing an array of oligonucleotides, the lengths and concentrations of the oligonucleotides being selected so that the nucleotide sequence can be determined after washing the matrix at a single temperature, said matrix formed by a multiplicity of gel portions according to the number of oligonucleotides in the array; wherein each gel portion contains one oligonucleotide of desired length and concentration from the array; said gel portions being separated from one another by interstitial spaces and the gel portions having a vertical height above the plane of the interstitial spaces of not more than 30 μm.

Claim 8

Independent Claim 119 words

A device for determining a nucleotide sequence, said device comprising: (a) a solid support; and (b) a matrix affixed to said support, said matrix containing an array of oligonucleotides, the lengths and concentrations of the oligonucleotides being selected so that the nucleotide sequence can be determined after washing the matrix at a single temperature, said matrix formed by a multiplicity of gel portions according to the number of oligonucleotides in the array; wherein each gel portion contains one oligonucleotide of desired length and concentration from the array; said gel portions being separated from one another by interstitial spaces and the gel portions having a vertical height above the plane of the interstitial spaces of not more than 30 μm.

Claim 9

Dependent Claim 58 words

The device according to claim 5 wherein each of said gel portions have one surface attached to the solid support while the opposite surface is directed outward; said outward-directed surface being a square wherein a length of a side is from 25 to 100 μm and a width of said interstices is double the length of the side.

Claim 10

Dependent Claim 20 words

The device according to claim 5 wherein each of said gel portions contains an oligonucleotide of a single selected length.

Claim 11

Independent Claim 117 words

A device for determining a nucleotide sequence said device comprising: (a) a solid support; and (b) a matrix affixed to said support, said matrix containing an array of oligonucleotides of a single length and a concentration selected so that the nucleotide sequence can be determined after washing the matrix at a single temperature, said matrix formed by a multiplicity of gel portions according to the number of oligonucleotides in the array, wherein each gel portion contains one oligonucleotide of desired length and concentration from the array; said gel portions being separated from one another by interstitial spaces and the gel portions having a vertical height above the plane of interstitial spaces of not more than 30 μm.

Claim 12

Independent Claim 117 words

A device for determining a nucleotide sequence said device comprising: (a) a solid support; and (b) a matrix affixed to said support, said matrix containing an array of oligonucleotides of a single length and a concentration selected so that the nucleotide sequence can be determined after washing the matrix at a single temperature, said matrix formed by a multiplicity of gel portions according to the number of oligonucleotides in the array, wherein each gel portion contains one oligonucleotide of desired length and concentration from the array; said gel portions being separated from one another by interstitial spaces and the gel portions having a vertical height above the plane of interstitial spaces of not more than 30 μm.

Claim 13

Independent Claim 84 words

A method for determining the sequence of a test nucleotide molecule: (a) providing an array of oligonucleotides wherein the array are present at concentrations effective to distinguish a perfect duplex from a one-base mismatch at a given temperature; (b) hybridizing the test nucleotide molecule to the oligonucleotides in the array; (c) washing the array at the given temperature; and (d) determining the oligonucleotides in the array which hybridized to the test nucleotide molecule to obtain information about the sequence of the test nucleotide molecule.

Claim 14

Independent Claim 84 words

A method for determining the sequence of a test nucleotide molecule: (a) providing an array of oligonucleotides wherein the array are present at concentrations effective to distinguish a perfect duplex from a one-base mismatch at a given temperature; (b) hybridizing the test nucleotide molecule to the oligonucleotides in the array; (c) washing the array at the given temperature; and (d) determining the oligonucleotides in the array which hybridized to the test nucleotide molecule to obtain information about the sequence of the test nucleotide molecule.

Annuity Stage	Window Opens	Payment Deadline	Surcharge Period Until	Estimated Fee	Status
3.5 Years (Year 4)	1999-09-03	2000-03-04	2000-09-03	$2000.00	Window Closed / Lapsed
7.5 Years (Year 8)	2003-09-03	2004-03-04	2004-09-03	$3760.00	Window Closed / Lapsed
11.5 Years (Year 12)	2007-09-03	2008-03-04	2008-09-03	$7700.00	Window Closed / Lapsed

Methods of DNA sequencing by hybridization based on optimizing concentration of matrix-bound oligonucleotide and device for carrying out same

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