Methods of preparing probe array by hybridation

• US 5,631,134 A
• Filed: 06/05/1995
• Issued: 05/20/1997
• Est. Priority Date: 11/06/1992
• Status: Expired due to Term

• Current Assignee: Trustees of Boston University
• Original Assignee: Trustees of Boston University
• Inventors: Cantor, Charles R.
• Primary Examiner(s): JONES, WILLIAM G
• Assistant Examiner(s): Tran, Paul B.
• Application Number:
  US08/462,704
• Time in Patent Office: 715 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 435/91.52, 935/77, 935/78, 536/22.1, 536/20.3
• US Class Current: 435/6.18
• 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 2219/00529 DNA chips
• CPC Class Codes: B01J 2219/0059 Sequential processes
• CPC Class Codes: B01J 2219/00605 the compounds being directly bound or immobilised to solid supports
• CPC Class Codes: B01J 2219/0061 The surface being organic
• CPC Class Codes: B01J 2219/00612 the surface being inorganic
• CPC Class Codes: B01J 2219/00626 Covalent
• CPC Class Codes: B01J 2219/00637 by coating it with another layer
• CPC Class Codes: B01J 2219/00648 by the use of solid beads
• CPC Class Codes: B01J 2219/00659 Two-dimensional arrays
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 2525/161 incorporating target specific and non-target specific sites
• CPC Class Codes: C12Q 2565/537 characterised by the capture oligonucleotide acting as a primer
• CPC Class Codes: C40B 40/06 Libraries containing nucleotides or polynucleotides, or derivatives thereof
• CPC Class Codes: C40B 80/00 Linkers or spacers specially adapted for combinatorial chemistry or libraries, e.g. traceless linkers or safety-catch linkers

Abstract

This invention is directed to methods for determining a nucleotide sequence of a nucleic acid using positional sequencing by hybridization, and to the creation of nucleic acids probes which may be used with these methods. This invention is also directed to diagnostic aids for analyzing the nucleic acid composition and content of biological samples, including samples derived from medical and agricultural sources.

Claims

1. A method for creating a nucleic acid probe array comprising the steps of:

a) hybridizing a plurality of single-stranded first nucleic acids to a plurality of longer, single-stranded second nucleic acids complementary to the first nucleic acids wherein each second nucleic acid contains a variable terminal nucleotide sequence to form probes each having a double-stranded portion and a single-stranded portion with the variable nucleotide sequence in the single-stranded portion;

b) hybridizing target nucleic acids to the probes;

c) ligating hybridized targets to the first nucleic acids of the probes;

d) isolating the second nucleic acids; and

e) hybridizing additional first nucleic acids to isolated second nucleic acids to form the nucleic acid probe array.

2. The method of claim 1 wherein each first nucleic acid is about 15 to about 25 nucleotides in length and each second nucleic acid is about 20 to about 30 nucleotides in length.

3. The method of claim 1 wherein the double-stranded portion contains an enzyme recognition site.

4. The method of claim 1 further comprising the steps of hybridizing ligated probes with an array of oligonucleotides containing variable nucleotide sequences and ligating hybridized oligonucleotides to the second nucleic acids of the probes.

5. The method of claim 1 further comprising the step of enzymatically extending the second nucleic acids after hybridization with targets using the targets as a template.

6. The method of claim 1 further comprising a step of fixing any of said nucleic acids to a solid support.

7. The method of claim 6 wherein the solid support is a plastic, a ceramic, a metal, a resin, a gel, or a membrane.

8. A method for creating a nucleic acid probe array comprising the steps of:

a) hybridizing a plurality of single-stranded first nucleic acids to a plurality of longer, single-stranded second nucleic acids complementary to the first nucleic acids wherein each second nucleic acid contains a variable terminal nucleotide sequence to form probes each having a double-stranded portion and a single-stranded portion with the variable nucleotide sequence in the single-stranded portion;

b) hybridizing target nucleic acids to the probes;

c) ligating hybridized targets to the first nucleic acids of the probes;

d) hybridizing ligated probes to an array of oligonucleotides that contain variable nucleotide sequences;

e) ligating hybridized oligonucleotides to the second nucleic acids of the probes;

f) isolating ligated second nucleic acids; and

g) hybridizing additional first nucleic acids to isolated second nucleic acids to form the nucleic acid probe array.

9. The method of claim 8 wherein each first nucleic acid is about 15 to about 25 nucleotides in length and each second nucleic acid is about 20 to about 30 nucleotides in length.

10. The method of claim 8 wherein the double-stranded portion contains an enzyme recognition site.

11. The method of claim 8 wherein the oligonucleotides are each about 4 to about 20 nucleotides in length.

12. The method of claim 8 which further comprising a step of fixing any of said nucleic acids to a solid support.

13. The method of claim 12 wherein the solid support is a plastic, ceramic, a metal, a resin, a gel, or a membrane.

17. The method of claim 10 further comprising a step of fixing any of said nucleic acids to a solid support.

18. The method of claim 17 wherein the solid support is a plastic, ceramic, a metal, a resin, a gel, or a membrane.

14. A method for creating a nucleic acid probe array comprising the steps of:

a) hybridizing a plurality of single-stranded first nucleic acids to a plurality of longer, single-stranded second nucleic acids complementary to the first nucleic acids wherein each second nucleic acid contains a variable terminal nucleotide sequence to form probes having a double-stranded portion and a single-stranded portion with the variable nucleotide sequence in the single-stranded portion;

b) hybridizing target nucleic acids to the probes;

c) ligating hybridized targets to the first nucleic acids of the probes;

d) enzymatically extending each second nucleic acid using the target as a template;

e) isolating extended second nucleic acids; and

f) hybridizing additional first nucleic acids to isolated second nucleic acids to form the nucleic acid probe array.

15. The method of claim 14 wherein each first nucleic acid is about 15 to about 25 nucleotides in length and each second nucleic acid is about 20 to about 30 nucleotides in length.

16. The method of claim 14 wherein the double-stranded portion contains an enzyme recognition site.