Solid phase sequencing of double-stranded nucleic acids

• US 6,436,635 B1
• Filed: 03/12/1996
• Issued: 08/20/2002
• Est. Priority Date: 11/06/1992
• Status: Expired due to Term

• Current Assignee: Sequenom Incorporated (Laboratory Corporation Of America Holdings), Trustees of Boston University
• Original Assignee: Boston University, Sequenom Incorporated (Laboratory Corporation Of America Holdings)
• Inventors: Fu, Dong-Jing, Smith, Cassandra L., Köster, Hubert, Cantor, Charles R.
• Primary Examiner(s): Horlick, Kenneth R.
• Application Number:
  US08/614,151
• Time in Patent Office: 2,352 Days
• Field of Search: 435/6, 536/23.1, 536/26.6
• US Class Current: 435/7.24
• 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 2219/0063 Other, e.g. van der Waals forces, hydrogen bonding
• 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 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/6872 involving mass spectrometry
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2525/161 incorporating target specific and non-target specific sites
• CPC Class Codes: C12Q 2565/501 being an array of oligonucleotides
• CPC Class Codes: C12Q 2565/537 characterised by the capture oligonucleotide acting as a primer
• CPC Class Codes: C12Q 2565/627 : being a mass spectrometer
• CPC Class Codes: C40B 40/06 : Libraries containing nucleotides or polynucleotides, or derivatives thereof

Abstract

This invention relates to methods for detecting and sequencing of target double-stranded nucleic acid sequences, to nucleic acid probes and arrays of probes useful in these methods, and to kits and systems which contain these probes. Useful methods involve hybridizing the nucleic acids or nucleic acids which represent complementary or homologous sequences of the target to an array of nucleic acid probes. These probe comprise a single-stranded portion, an optional double-stranded portion and a variable sequence within the single-stranded portion. The molecular weights of the hybridized nucleic acids of the set can be determined by mass spectroscopy, and the sequence of the target determined from the molecular weights of the fragments. Nucleic acids whose sequences can be determined include nucleic acids in biological samples such as patient biopsies and environmental samples. Probes may be fixed to a solid support such as a hybridization chip to facilitate automated determination of molecular weights and identification of the target sequence.

Claims

1. A method for sequencing a target nucleic acid comprising the steps of:

a) providing a set of partially single-stranded nucleic acid fragments, wherein each fragment contains a sequence that corresponds to a sequence of the target;

b) hybridizing the single-stranded portions of the fragments to single-stranded portions of a set of partially double-stranded nucleic acid probes to form a set of complexes, and for each complex;

i) ligating a single strand of the fragment to an adjacent single strand of the probe; and

ii) extending the unligated strand of the complex by strand-displacement polymerization using the ligated strand as a template; and

c) determining the sequence of the target.

2. The method of claim 1 wherein the target is between about 10 to about 1,000 nucleotides in length.

3. The method of claim 1 wherein the set of partially single-stranded fragments is a product of a polymerase chain reaction or an endonuclease digestion.

4. The method of claim 1 wherein each set of nucleic acids comprises greater than about 10⁴ different members.

5. The method of claim 1 wherein the single-stranded portion of each probe contains a variable sequence.

6. The method of claim 1 wherein the fragments or the probes are attached to a solid support.

7. The method of claim 1 wherein the nucleic acids are DNA, RNA, PNA or modifications or combinations thereof.

8. The method of claim 1 wherein the fragments or the probes are dephosphorylated by treatment with a phosphatase prior to hybridization.

9. The method of claim 1 wherein each probe is between about 10 to about 50 nucleotides in length.

10. The method of claim 1 wherein the double-stranded regions of the probes contain the same sequence for each probe of the set.

11. The method of claim 1 wherein polymerization incorporates mass-modifying nucleotides into the extended strand.

12. The method of claim 1 wherein polymerization incorporates dNTPs and ddNTPs into the extended strand.

13. The method of claim 12 wherein a plurality of extended strands comprise a nested set.

14. The method of claim 1 wherein a plurality of extended strands comprise about 0.1 femtomole to about 1.0 nanomole of nucleic acid.

15. The method of claim 1 wherein the sequence is determined by polyacrylamide electrophoresis, capillary electrophoresis or mass spectrometry.

16. A method for sequencing a target nucleic acid comprising the steps of:

a) providing a set of partially single-stranded nucleic acid fragments, wherein each fragment contains a sequence that corresponds to a sequence of the target;

b) hybridizing the single-stranded portions of the fragments to single-stranded portions of a set of partially double-stranded nucleic acid probes to form a set of complexes, and for each complex;

i) ligating a single strand of the fragment to an adjacent single strand of the probe; and

ii) extending the unligated strand of the complex by strand-displacement polymerization using the ligated strand as a template and mass-modifying the extended strand;

c) determining the molecular weights of the extended strands; and

d) determining the sequence of the target from the molecular weights of the extended strands.

17. The method of claim 16 wherein the extended strand contains mass-modifying functionalities.

18. The method of claim 17 wherein at least one mass-modifying functionality is coupled to a heterocyclic base.

19. The method of claim 17 wherein at least one mass-modifying functionality is coupled to a sugar moiety.

20. The method of claim 17 wherein at least one mass-modifying functionality is coupled to a phosphate group.

21. The method of claim 17 wherein the mass-modifying functionality is a chemical moiety that does not interfere with hydrogen bonding for base-pair formation.

22. The method of claim 17 wherein the mass-modifying functionality is coupled to a purine at position C2, N3, N7 or C8, or a deazapurine at position N7 or N9.

23. The method of claim 17 wherein the mass-modifying functionality is coupled to a pyrimidine at position C5 or C6.

24. The method of claim 17, wherein;

the mass-modifying functionality is selected from the group consisting of deuterium, F, Cl, Br, I, SiR₃, Si(CH₃)₃, Si(CH₃)₂(C₂H₅), Si(CH₃)(C₂H₅)₂, Si(C₂H₅)₃, (CH₂)_nCH₃, (CH₂)_nNR₂, CH₂CONR₂, (CH₂)_nOH, CH₂F, CHF₂, and CF₃;

n is an integer; and

R is selected from the group consisting of —

H, deuterium and alkyls, alkoxys and aryls of 1-6 carbon atoms, polyoxymethylene, monoalkylated polyoxymethylene, polyethylene imine, polyamide, polyester, alkylated silyl, heterooligo/polyaminoacid and polyethylene glycol.

25. The method of claim 17, wherein;

the mass-modifying functionality is —

N₃ or —

XR;

X is selected from the group consisting of —

O—

, —

NH—

, —

NR—

, —

S—

, —

NHC(S)—

, —

OCO(CH₂)_nCOO—

, —

NHCO(CH₂)_nCOO—

, —

OSO₂O—

, —

OCO(CH₂)_n—

, —

NHC(S)NH—

, —

OCO(CH₂)_nS—

, —

OCO(CH₂)S—

, —

NC₄O₂H₂S—

, —

OPO(O-alkyl)—

, and OP(O-alkyl)—

;

n is an integer from 1 to 20; and

R is selected from the group consisting of —

H, deuterium and alkyls, alkoxys and aryls of 1-6 carbon atoms, polyoxymethylene, monoalkylated polyoxymethylene, polyethylene imine, polyamide, polyester, alkylated silyl, heterooligo/polyaminoacid and polyethylene glycol.

26. The method of claim 16 wherein the extended strand is mass-modified by thiolation.

27. The method of claim 26 wherein thiolation is performed by treating said extended strand with a Beaucage reagent.

28. The method of claim 16 wherein the extended strand is mass-modified by alkylation.

29. The method of claim 28 wherein alklyation is performed by treating said extended strand with iodoacetamide.

30. The method of claim 16 further comprising the step of removing alkali cations from said mass-modified extended strand.

31. The method of claim 30 wherein alkali cations are removed by ion exchange.

40. The method of claim 16, wherein the molecular weights of the extended strands are determined by mass spectrometry.

41. The method of claim 40, wherein the mass spectrometry includes fast atom bombardment, plasma desorption, matrix-assisted laser desorption/ionization, electrospray, photochemical release, electrical release, droplet release, resonance ionization or a combination thereof.

42. The method of claim 40, wherein the mass spectrometry format includes time of flight with reflection, time of flight without reflection, electrospray, Fourier transform, ion trap, resonance ionization, ion cyclotron resonance or a combination thereof.

43. The method of claim 40, wherein the molecular weights of two or more extended strands are determined simultaneously.

32. A method for sequencing a target nucleic acid comprising the steps of:

a) providing a set of partially single-stranded nucleic acid fragments wherein each fragment contains a sequence that corresponds to a sequence of the target;

b) hybridizing the single-stranded portions of the fragments to single-stranded portions of a set of partially double-stranded nucleic acid probes to form a set of complexes wherein each probe contains a variable sequence within the single-stranded region, and for each complex;

i) ligating a single strand of the fragment to an adjacent single strand of the probe; and

ii) extending the unligated strand of the complex by strand-displacement polymerization using the ligated strand as a template;

c) determining the molecular weights of the extended strands; and

d) determining the sequence of the target from the molecular weights of the extended strands.

33. The method of claim 32 wherein one set of nucleic acids is attached to a solid support.

34. The method of claim 33 wherein the solid support is selected from the group consisting of plates, beads, microbeads, whiskers, combs, hybridization chips, membranes, single crystals, ceramics and self-assembling monolayers.

35. The method of claim 33 wherein the probes are conjugated with biotin or a biotin derivative and the solid support is conjugated with avidin, streptavidin or a derivative thereof.

36. The method of claim 33 further comprising a spacer between the array and the solid support.

37. The method of claim 36 wherein the spacer is selected from the group consisting of oligopeptides, oligonucleotides, oligopolyamides, oligoethyleneglycerol, oligoacrylamides, alkyl chains of between about 6 to about 20 carbon atoms, and combinations thereof.

38. The method of claim 33 wherein the attachment is a cleavable attachment.

39. The method of claim 38 wherein the cleavable attachment is cleavable by heat, an enzyme, a chemical agent or electromagnetic radiation.

44. The method of claim 32, wherein the molecular weights of the extended strands are determined by mass spectrometry.

45. The method of claim 44, wherein the mass spectrometry includes fast atom bombardment, plasma desorption, matrix-assisted laser desorption/ionization, electrospray, photochemical release, electrical release, droplet release, resonance ionization or a combination thereof.

46. The method of claim 44, wherein the mass spectrometry includes time of flight with reflection, time of flight without reflection, electrospray, Fourier transform, ion trap, resonance ionization, ion cyclotron resonance or a combination thereof.

47. The method of claim 44, wherein the molecular weights of two or more extended strands are determined simultaneously.