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

• US 5,552,270 A
• Filed: 11/09/1992
• Issued: 09/03/1996
• Est. Priority Date: 03/18/1991
• Status: Expired due to Term

• Current Assignee: Institut Molekulyarnoi Biologii IMVA Engelgardta Rossiiskoi Akademii NAUK
• Original Assignee: Institut Molekulyarnoi Biologii Imeni V.A.
• Inventors: Ershov, Gennady M., Ivanov, Igor B., Khrapko, Konstantin R., Khorlin, Alexandr A., Lysov, Jury P., Mirzabekov, Andrei D., Florentiev, Vladimir L.
• Primary Examiner(s): Horlick, Kenneth R.
• Application Number:
  US07/949,541
• Time in Patent Office: 1,394 Days
• Field of Search: 422/55, 422/56, 422/57, 422/58, 422/68.1, 436/94, 436/501, 935/77, 935/78, 435/6
• US Class Current: 435/6.1
• CPC Class Codes: B01J 19/0046 Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays synthesis methods per se C40B50/00
• CPC Class Codes: B01J 2219/00527 Sheets
• CPC Class Codes: B01J 2219/0059 Sequential processes
• CPC Class Codes: B01J 2219/00596 Solid-phase processes
• CPC Class Codes: B01J 2219/00605 the compounds being directly bound or immobilised to solid supports
• CPC Class Codes: B01J 2219/00612 the surface being inorganic
• CPC Class Codes: B01J 2219/00621 by physical means, e.g. trenches, raised areas
• CPC Class Codes: B01J 2219/00626 Covalent
• CPC Class Codes: B01J 2219/00641 the porous medium being continuous, e.g. porous oxide substrates
• CPC Class Codes: B01J 2219/00644 the porous medium being present in discrete locations, e.g. gel pads
• CPC Class Codes: B01J 2219/00659 Two-dimensional arrays
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2527/107 Temperature of melting, i.e. Tm
• CPC Class Codes: C12Q 2527/143 Concentration of primer or probe
• CPC Class Codes: C40B 40/06 Libraries containing nucleotides or polynucleotides, or derivatives thereof
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

A method for sequencing DNA by hybridization that includes the following steps: forming an array of oligonucleotides at such concentrations that either ensure the same dissociation temperature for all fully complementary duplexes or allows hybridization and washing of such duplexes to be conducted at the same temperature; hybridizing said oligonucleotide array with labeled test DNA; washing in duplex dissociation conditions; identifying single-base substitutions in the test DNA by analyzing the distribution of the dissociation temperatures and reconstructing the DNA nucleotide sequence based on the above analysis. A device for carrying out the method comprises a solid substrate and a matrix rigidly bound to the substrate. The matrix contains the oligonucleotide array and consists of a multiplicity of gel portions. Each gel portion contains one oligonucleotide of desired length. The gel portions are separated from one another by interstices and have a thickness not exceeding 30 μm.

Claims

1. 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).

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

3. 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.

4. 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).

5. 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 μ

m.

6. 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 μ

m and a width of said interstices is double the length of the side.

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

8. 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 μ

m.

9. 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.