Positional sequencing by hybridization

• US 5,503,980 A
• Filed: 10/17/1994
• Issued: 04/02/1996
• 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, W. Gary
• Assistant Examiner(s): TRAN, PAUL
• Application Number:
  US08/322,526
• Time in Patent Office: 533 Days
• Field of Search: 435/6, 435/91.1, 435/91.52, 935/77, 935/78, 536/23.1, 536/24.32
• US Class Current: 435/6.16
• 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 determining a nucleotide sequence of a nucleic acid target by hybridization comprising the steps of:

(a) creating a set of nucleic acid probes wherein each probe is 14-50 nucleotides in length and has a double stranded portion, a single stranded portion, and a variable sequence within the single stranded portion which is determinable;

(b) hybridizing the target which is at least partly single stranded to one or more of the nucleic acid probes; and

(c) determining the nucleotide sequence of the target which is hybridized to the single stranded portion of any probe.

3. The method of claim 1 wherein the nucleic acid target is selected from the group consisting of DNA, RNA and PNA.

4. The method of claim 1 wherein the nucleic acid probe is selected from the group consisting of DNA, RNA and PNA.

5. The method of claim 1 wherein the variable nucleotide sequence within the single stranded portion of the probe is about 4-20 nucleotides in length.

6. The method of claim 1 wherein the double stranded portion of each probe is constant and is about 10-30 nucleotides in length.

7. The method of claim 1 wherein the target is about 30-300 nucleotides in length.

8. The method of claim 1 wherein the set of nucleic acid probes is fixed to a solid support.

9. The method of claim 8 wherein the solid support is a plastic, a ceramic, a metal, a resin, a gel, or a membrane.

10. The method of claim 8 wherein the solid support is a two-dimensional or three-dimensional matrix with multiple probe binding sites.

11. The method of claim 10 wherein the two-dimensional or three-dimensional matrix is a hybridization chip.

12. The method of claim 1 wherein the target is labeled with a detectable label.

13. The method of claim 12 wherein the detectable label is a radioisotope, a stable isotope, an enzyme, a fluorescent chemical, a luminescent chemical, a chromatic chemical, a metal, an electric charge, or a spatial chemical.

14. The method of claim 12 wherein the target is labeled with a first detectable label at a terminal site and a second different detectable label at an internal site.

15. The method of claim 14 wherein the first and second detectable labels are chromatic or fluorescent chemicals.

16. The method of claim 14 wherein the first and second labels are detectable by mass spectrometry.

2. A method for determining a nucleotide sequence of a nucleic acid target by hybridization comprising the steps of:

(a) creating a set of nucleic acid probes wherein each probe is 14-50 nucleotides in length and has a double stranded portion, a single stranded portion, and a variable sequence within the single stranded portion which is determinable;

(b) hybridizing the target which is at least partly single stranded to one or more of the nucleic acid probes;

(c) ligating the hybridized target to the probe; and

(d) determining the nucleotide sequence of the target which is hybridized to the single stranded portion of any probe.

17. A method for determining a nucleotide sequence of a target nucleic acid by hybridization comprising the steps of:

(a) creating a set of nucleic acid probes wherein each probe is 14-50 nucleotides in length and has a double stranded portion at a 3'"'"'-terminus, a single stranded portion at a 5'"'"'-terminus, and a variable sequence within the single stranded portion which is determinable;

(b) hybridizing the target nucleic acid which is at least partly single stranded to the set;

(c) enzymatically extending a strand of the probe using the hybridized target as a template; and

(d) determining the nucleotide sequence of the single stranded portion of the target nucleic acid.

19. The method of claim 17 wherein the probe is enzymatically extended by DNA polymerase, using one or more dideoxynucleotide triphosphates and one or more deoxynucleotides triphosphates.

20. The method of claim 17 wherein the probe is enzymatically extended by DNA polymerase using one dideoxynucleotide triphosphate.

21. The method of claim 17 wherein the probe has a detectable label.

22. The method of claim 21 wherein the detectable label is a radioisotope, a stable isotope, an enzyme, a fluorescent chemical, a luminescent chemical, a chromatic chemical, a metal, a electric charge, or a spatial structure.

23. The method of claim 17 wherein the target is labeled with a first detectable label at a terminal site and a second different detectable label at an internal site.

24. The method of claim 23 wherein the first and second detectable labels are chromatic or fluorescent chemicals.

25. The method of claim 23 wherein the first and second labels are detectable by mass spectrometry.

18. A method for determining a nucleotide sequence of a target nucleic acid by hybridization comprising the steps of:

(a) creating a set of nucleic acid probes wherein each probe is 14-50 nucleotides in length and has a double stranded portion at a 3'"'"'-terminus, a single stranded portion at a 5'"'"'-terminus, and a variable sequence within the single stranded portion which is determinable;

(b) hybridizing the target nucleic acid which is at least partly single stranded to the set;

(c) ligating the hybridized target to the probe;

(d) enzymatically extending a strand of the probe using the hybridized target as a template; and

(e) determining the nucleotide sequence of the single stranded portion of the target nucleic acid.

26. A method for determining a nucleotide sequence of a target by hybridization comprising the steps of:

(a) creating a set of nucleic acid probes wherein each probe is about 14-50 nucleotides in length and has a double-stranded portion, a single-stranded portion, and a variable nucleotide sequence within the single-stranded portion which is determinable;

(b) cleaving a plurality of nucleic acid targets to form fragments of various lengths which are at least partly single-stranded;

(c) hybridizing the single-stranded region of the fragments with the single-stranded region of the probes;

(d) identifying the nucleotide sequences of the hybridized portions of the fragments; and

(e) comparing the identified nucleotide sequences to determine the nucleotide sequence of the target.

28. The method of claim 26 wherein cleaving occurs by enzymatic, chemical or physical means.

29. The method of claim 26 wherein the set of nucleic acid probes is fixed to a solid support.

30. The method of claim 29 wherein the solid support is a plastic, a ceramic, a metal, a resin, a gel, or a membrane.

31. The method of claim 29 wherein the solid support is a two-dimensional or three-dimensional matrix with multiple probe binding sites.

32. The method of claim 26 wherein the target nucleic acid has a detectable label.

33. The method of claim 32 wherein the detectable label is a radioisotope, a stable isotope, an enzyme, a fluorescent chemical, a luminescent chemical, a chromatic chemical, a metal, a electric charge, or a spatial structure.

34. The method of claim 32 wherein the target nucleic acid has a first detectable label at a terminal site and a second detectable label at an internal site.

35. The method of claim 34 wherein the first and second detectable labels are chromatic or fluorescent chemicals.

36. The method of claim 34 wherein the first and second labels are detectable by mass spectrometry.

27. A method for determining a nucleotide sequence of a target by hybridization comprising the steps of:

(a) creating a set of nucleic acid probes wherein each probe is about 14-50 nucleotides in length and has a double-stranded portion, a single-stranded portion, and a variable nucleotide sequence within the single-stranded portion which is determinable;

(b) cleaving a plurality of nucleic acid targets to form fragments of various lengths which are at least partly single-stranded;

(c) hybridizing the single-stranded region of the fragments with the single-stranded region of the probes;

(d) ligating the hybridized fragments to the probes;

(e) identifying the nucleotide sequences of the hybridized portions of the fragments; and

(f) comparing the identified nucleotide sequences to determine the nucleotide sequence of the target.

37. A method for determining a nucleotide sequence within a nucleic acid fragment comprising the steps of:

(a) labeling the nucleic acid fragment with a first detectable label at a terminal site and with a second detectable label in a plurality of internal sites;

(b) cleaving the labeled nucleic acid fragment into portions;

(c) aligning the relative positions of said portions to the nucleic acid fragment by determining the ratio of the first detectable label to the second detectable label; and

(d) determining the nucleotide sequence of said portions to determine the nucleotide sequence within said nucleic acid fragment.