Double ended sequencing

• US 7,244,567 B2
• Filed: 01/28/2004
• Issued: 07/17/2007
• Est. Priority Date: 01/29/2003
• Status: Expired due to Fees

• Current Assignee: 454 Life Sciences Corporation (Roche Holding AG)
• Original Assignee: 454 Life Sciences Corporation (Roche Holding AG)
• Inventors: Weiner, Michael P., Rothberg, Jonathan M., Lohman, Kenton L., Leamon, John H., Srinivasan, Maithreyan, Chen, Yi-Ju, Ronan, Michael T.
• Primary Examiner(s): Fredman; Jeffrey
• Assistant Examiner(s): Thomas; David C.
• Application Number:
  US10/768,729
• Publication Number:
  US 20060134633A1
• Time in Patent Office: 1,266 Days
• Field of Search: None
• US Class Current: 435/6.11
• CPC Class Codes: B01L 2300/0636 Integrated biosensor, microarrays
• CPC Class Codes: B01L 2300/0819 Microarrays; Biochips
• CPC Class Codes: B01L 2300/0877 Flow chambers
• CPC Class Codes: B01L 3/5027 by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip B01L3/5023 takes precedence; micromixers B01F33/30; microreactors for synthesis B01J19/0093; microcapillary devices in general B81B1/00
• CPC Class Codes: B01L 3/502707 characterised by the manufacture of the container or its components by shaping or joining plastic parts B29C59/00, B29C65/00; by laminating B32B37/00; manufacture of microstructural devices in general B81C
• CPC Class Codes: B01L 3/502715 characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
• CPC Class Codes: B01L 3/5085 for multiple samples, e.g. microtitration plates
• CPC Class Codes: B01L 7/52 with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples amplification or hybridisation processes per se C12Q1/68; controlling sequential reactions for synthesis B01J19/0046
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C12N 15/1075 by coupling phenotype to genotype, not provided for in other groups of this subclass
• CPC Class Codes: C12N 15/1093 General methods of preparing gene libraries, not provided for in other subgroups
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/6865 Promoter-based amplification, e.g. nucleic acid sequence amplification [NASBA], self-sustained sequence replication [3SR] or transcription-based amplification system [TAS]
• CPC Class Codes: C12Q 1/6867 Replicase-based amplification, e.g. using Q-beta replicase
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2525/186 incorporating a non-extendable or blocking moiety
• CPC Class Codes: C12Q 2527/125 : Specific component of sample, medium or buffer
• CPC Class Codes: C12Q 2531/113 : PCR
• CPC Class Codes: C12Q 2531/125 : Rolling circle
• CPC Class Codes: C12Q 2535/119 : Double strand sequencing
• CPC Class Codes: C12Q 2537/143 : Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2537/149 : Sequential reactions
• CPC Class Codes: C12Q 2547/101 : by confinement to a single tube/container
• CPC Class Codes: C12Q 2547/107 : Use of permeable barriers, e.g. waxes
• CPC Class Codes: C12Q 2563/149 : Particles, e.g. beads
• CPC Class Codes: C12Q 2565/119 : based on extraction of label to an organic phase, i.e. partitioning of label between different organic phases
• CPC Class Codes: C12Q 2565/301 : Pyrophosphate (PPi)
• CPC Class Codes: C12Q 2565/537 : characterised by the capture oligonucleotide acting as a primer
• CPC Class Codes: G01N 2021/6484 : Optical fibres
• CPC Class Codes: G01N 21/253 : for batch operation, i.e. multisample apparatus analytical automats G01N35/00
• CPC Class Codes: G01N 21/6428 : Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" in vivo A61B5/00; immunoassay G01N33/53
• CPC Class Codes: G01N 21/6452 : Individual samples arranged in a regular 2D-array, e.g. multiwell plates
• CPC Class Codes: G01N 21/6458 : Fluorescence microscopy fluorescence microscopes per se G02B21/0076 and G02B21/16
• CPC Class Codes: G01R 33/1269 : of molecules labeled with magnetic beads magnetic particles for bio assay G01N33/54326
• CPC Class Codes: Y02P 20/582 : Recycling of unreacted starting or intermediate materials
• CPC Class Codes: Y10T 436/143333 : Saccharide [e.g., DNA, etc.]

Abstract

This invention relates to methods of sequencing DNA. More specifically, this invention relates to methods of sequencing both the sense and antisense strands of DNA through the use of blocked and unblocked sequencing primers. In brief, these methods include the steps of annealing an unblocked primer to a first strand of nucleic acid; annealing a second blocked primer to a second strand of nucleic acid; elongating the nucleic acid along the first strand with a polymerase; terminating the first sequencing primer; deblocking the second primer; and elongating the nucleic acid along the second strand.

Claims

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

(a) hybridizing two or more sequencing primers to one or a plurality of single strands of the nucleic acid molecule wherein all the primers except for one are reversibly blocked primers;

(b) incorporating at least one base onto the nucleic acid molecule by polymerase elongation from an unblocked primer;

(c) preventing further elongation of said unblocked primer

(d) deblocking one of the reversibly blocked primers into an unblocked primer;

(e) repeating steps (b) to (d) until at least one of the reversibly blocked primers are deblocked and used for determining a sequence.

2. The method of claim 1, wherein said step (c) of preventing further elongation comprises

(a) completing the elongation from the unblocked primer with polymerase and dNTPs;

or

(b) terminating the elongation with polymerase in a manganese containing buffer, dNTPs, and at least one ddNTP;

or

(c) terminating the elongation chemically.

3. The method of claim 1 further comprising the step of removing said polymerase, dNTPs, and ddNTPs before said step (d).

4. The method of claim 1 wherein at least one reversibly blocked primer is blocked by a chemical moiety selected from the group consisting of a PO₄ group, a thio group, and a phosphorothiol group.

5. The method of claim 1 wherein at least one reversibly blocked primer has a 3′

mismatched end that can be deblocked by contacting said primer with an exonuclease.

6. The method of claim 1 wherein at least one reversibly blocked primer has one or more noncomplementary bases that forms a loop and does not hybridize to the nucleic acid molecule, wherein said one or more bases is not at the 5′

or 3′

end of said reversibly blocked primer, and wherein said reversibly blocked primer comprises a dideoxy nucleotide at its 3′

end.

7. The method of claim 6 wherein said at least one reversibly blocked primer is unblocked by endonuclease digestion of said one or more noncomplementary bases forming a nick at said one or more noncomplementary base.

8. The method of claim 7 wherein step (b) comprises polymerase elongation at said nick by a strand-displacing polymerase.

9. The method of claim 1 wherein at least one reversibly blocked primer has a sequence of 5′

-NUX-3′

wherein N represents an oligonucleotide sequence of any length, U is uracil, and X is a dideoxy-nucleotide.

10. The method of claim 9 wherein said reversibly blocked primer is unblocked by Uracil DNA glycosylase and AP endonuclease to generate an unblocked primer with an extendable 3′

end.

11. The method of claim 1 at least one reversibly blocked primer has a sequence of 5′

-NYZ-3′

wherein N represents an oligonucleotide sequence of any length, Y is a modified nucleotide, and Z represents a single nucleotide base; and

wherein said modified nucleotide can be deblocked by formamidopyrimidine (fapy)-DNA glycosylase.

12. The method of claim 11 wherein said modified base is selected from the group consisting of 8-oxoguanine, 8-oxoadenine, fapy-guanine, methyl-fapy-guanine, fapy-adenine, aflatoxin B₁-fapy-guanine, 5-hydroxy cytosine and 5-hydroxy-uracil.

13. The method of claim 1 wherein the nucleic acid molecule is a genomic DNA, cDNA, or episomal DNA.

14. The method of claim 1 wherein at least one sequencing primer hybridizes to a sense strand of the nucleic acid and at least one sequencing primer hybridizes to an antisense strand of the nucleic acid molecule in step (a).

15. The method of claim 1 wherein the polymerase elongation is between 1 and 250 bases.

16. The method of claim 1 wherein said method is performed in a reaction vessel selected from the group consisting of a test tube, a reaction chamber of a PicoTiter plate, a reaction chamber of an array, and a microencapsulated reaction chamber of a water-in-oil emulsion.

17. The method of claim 1 wherein the nucleic acid molecule is between 100 to 1000 bp in length.

18. The method of claim 1 wherein at least one said strand of nucleic acid molecule or at least one said primers is attached to a solid support.

19. The method of claim 1 wherein at least one strand of said nucleic acid molecule is linked to a solid support.

20. The method of claim 18, wherein at least one said primer is immobilized on a solid support to form an immobilized primer and said at least one strand is linked to a solid support by hybridization with said immobilized primer.

21. The method of claim 20 wherein the solid support is a spherical mobile solid support.

22. The method of claim 1 wherein at least one primer comprise a detectable label.

23. The method of claim 1 wherein the method of determining a sequence is pyrophosphate sequencing or Sanger sequencing.

24. The method of claim 1 wherein the deblocking step comprises contacting a reversibly blocked primer with an agent to remove a PO₄.group on said reversibly blocked primer.

25. The method of claim 24 wherein said agent is selected from the group consisting of polynucleotide kinase and alkaline phosphatase.

26. The method of claim 1 wherein said polymerase is devoid of 3′

to 5′

exonuclease activity.

27. The method of claim 1 wherein said method determines a first nucleic acid sequence proximate to one end of said nucleic acid molecule and a second nucleic acid sequence proximate to a second end of said nucleic acid molecule.

28. A method of sequencing a nucleic acid molecule comprising:

(a) hybridizing a first unblocked sequencing primer to a first strand of the nucleic acid molecule;

(b) hybridizing a second blocked sequencing primer to a second strand of the nucleic acid molecule;

(c) incorporating at least one base onto said first strand by extending said first unblocked primer with a polymerase;

(d) preventing further elongation of said unblocked primer;

(e) deblocking the second sequencing primer; and

(f) incorporating at least one base onto said second strand by extending said second primer with a polymerase;

wherein steps (a) and (b) are performed in any order or simultaneously.

29. The method of claim 28, wherein said step (d) of preventing further elongation comprises

(a) completing the elongation from the unblocked primer with polymerase and dNTPs;

or

(b) terminating the elongation with polymerase in a manganese containing buffer, dNTPs, and at least one ddNTP;

or

(c) terminating the elongation chemically.

30. The method of claim 29 further comprising the step of removing said polymerase, dNTPs, and ddNTPs after said preventing step.

31. The method of claim 28 wherein said second primer is blocked by a chemical moiety selected from the group consisting of a PO₄ group, a thio group, and a phosphorothiol group.

32. The method of claim 28 wherein said method determines at least a first nucleic acid sequence proximal to a first end of said nucleic acid molecule and determines a second nucleic acid sequence proximal to a second end of said nucleic acid molecule.

33. A method of determining a molecular haplotype of a DNA sample at multiple loci comprising the steps of:

(a) hybridizing 2 or more sequencing primers adjacent to a plurality of loci in a DNA sample wherein all the primers except for one are reversibly blocked primers and wherein each locus contains a nucleic acid sequence that determines a haplotype;

(b) determining a haplotype at one locus by polymerase elongation from an unblocked primer;

(c) preventing further elongation of said unblocked primer;

(d) deblocking one of the reversibly blocked primers into an unblocked primer;

(e) repeating steps (b) to (d) until all the reversibly blocked primers are deblocked and used for determining a molecular haplotype.

34. The method of claim 33, wherein said step (c) of preventing further elongation comprises

(a) completing the elongation from the unblocked primer with polymerase and dNTPs;

or

(b) terminating the elongation with polymerase in a manganese containing buffer, dNTPs, and at least one ddNTP;

or

(c) terminating the elongation chemically.

35. The method of claim 34 further comprising the step of removing said polymerase, dNTPs, and ddNTPs after said preventing step.