Methods and means for nucleic acid sequencing

• US 20060147935A1
• Filed: 02/09/2004
• Published: 07/06/2006
• Est. Priority Date: 02/12/2003
• Status: Abandoned Application

• Current Assignee: Genizon Svenska AB
• Original Assignee: Genizon Svenska AB
• Inventors: Linnarsson, Sten
• Application Number:
  US10/544,987
• Publication Number:
  US 20060147935A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2523/319 Photocleavage, photolysis, photoactivation
• CPC Class Codes: C12Q 2535/101 Sanger sequencing method, i.e. oligonucleotide sequencing using primer elongation and dideoxynucleotides as chain terminators
• CPC Class Codes: C12Q 2537/149 Sequential reactions

Abstract

Nucleic acid sequencing-by-synthesis. Primed synthesis of a second strand complementary to a template strand in repeated sets of steps, each step comprising providing one or more of the possible nucleotide complementarity classes for incorporation into the synthesized strand, and each set of steps comprising providing all four possible nucleotide complementarity classes. Three of the four possible nucleotide complementarity classes may first be provided for incorporation into the synthesized strand, then separately the fourth nucleotide complementarity class alone. Also, a DNA molecule consisting of a stem portion and first and second loop portions, wherein the stem portion consists of a first strand and a second strand, wherein the first strand and second strand are equal in length, complementary and annealed together, wherein the first loop portion joins the 3′ end of the first strand to the 5′ end of the second strand and the second loop portion joins the 3′ end of the second strand to the 5′ end of the first strand so the DNA molecule has no free 5′ or 3′ ends, and uses thereof, especially in sequencing.

Claims

1. A method of determining sequence and/or base composition information for a nucleic acid, the method comprising:

(i) providing a nucleic acid comprising a first strand that comprises a nucleic acid template, wherein a free 3′

end of a nucleic acid strand annealed to the first strand allows for elongation of a strand of nucleic acid complementary to the nucleic acid template by template sequence-dependent incorporation of nucleotides into the strand of nucleic acid complementary to the nucleic acid template by a template-dependent nucleic acid polymerase;

(ii) performing a set of one or more steps, which set of one or more steps is cycled a desired number of times or performed in combination with other sets of one or more steps to elongate the strand of nucleic acid complementary to the nucleic acid template allowing for information indicative of base composition or sequence of the nucleic acid to be obtained,

wherein a step comprises;

(a) providing, in the presence of;

the nucleic acid comprising a first strand that comprises a nucleic acid template,

said free 3′

end of a nucleic acid strand annealed to the first strand of the nucleic acid template, and

a template-dependent nucleic acid polymerase;

nucleotides selected from one, two, three or four nucleotide complementarity classes for template-dependent incorporation by the nucleic acid polymerase of the nucleotides into the strand of nucleic acid complementary to the nucleic acid template, wherein each of said nucleotides is a natural nucleotide or a nucleotide analog capable of template-dependent incorporation by a nucleic acid polymerase into a nucleic acid strand at a free 3′

end of the nucleic acid strand, and within each said nucleotide complementarity class the nucleotides and nucleotide analogs are complementary to one of Adenosine (A), Cytosine (C), Thymine (T) and Guanine (G);

and

(b) removing or inactivating unincorporated nucleotides;

and

wherein within a set of steps

nucleotides selected from all four nucleotide complementarity classes are provided and available for template-dependent incorporation,

in at least one step nucleotides selected from more than one, optionally two, three or four, nucleotide complementarity classes are provided and available for template-dependent incorporation, and the nucleotides in at least one of the nucleotide complementarity classes, if incorporated into the strand of nucleic acid complementary to the nucleic acid template, allow further elongation of the strand of nucleic acid complementary to the nucleic acid template, and

optionally no nucleotide complementarity class is provided in more than one step;

and

wherein if nucleotides selected from all four complementarity classes are provided in one step then the nucleotides in one, two or three of the nucleotide complementarity classes, if incorporated into the strand of nucleic acid complementary to the nucleic acid template, prevent further elongation of the strand of nucleic acid complementary to the nucleic acid template and all copies present if multiple copies are present;

(iii) performing multiple sets of said steps, cycling sets of steps and/or performing sets of steps in combination with different sets of steps;

(iv) determining the nature of and/or quantity of nucleotides incorporated into the strand of nucleic acid complementary to the nucleic acid template in at least one set of steps by determining the nature and/or quantity of nucleotides incorporated into the strand of nucleic acid complementary to the nucleic acid template in at least one step in each set for which the nature and/or quantity of nucleotides incorporated is determined for the set.

2. A method according to claim 1 wherein within a set of steps nucleotides selected from three or two of the nucleotide complementarity classes are provided in a first step and nucleotides taken from the remaining one or two nucleotide complementarity classes are provided in a second step.

3. A method according to claim 2 comprising determining the quantity of the nucleotide or nucleotides incorporated in the first or second step in sets of steps for which the nature and/or quantity of nucleotides incorporated is determined.

4. A method according to claim 3 comprising determining the quantity of nucleotides incorporated in each step in sets for which the quantity of nucleotides incorporated is determined.

5. A method according to claim 4 wherein within a set of steps three nucleotides are provided in a first step and one nucleotide is provided in a second step.

6. A method according to claim 5 comprising determining the nature and quantity of nucleotides incorporated in the first step.

7. A method according to claim 2 wherein the nucleotides provided in the first step are labeled, each differently.

8. A method according to claim 2 wherein a nucleotide provided in the second step is labeled.

9. A method according to claim 1 wherein the four nucleotides complementary to A, C, T and G are labeled, each differently.

10. A method according to claim 7, wherein a nucleotide is labeled fluorescently.

11. A method according to claim 7 wherein a label of a nucleotide is disabled when the nucleotide is incorporated into the strand of nucleic acid complementary to the nucleic acid template.

12. A method according to claim 7 wherein a label of a nucleotide is cleaved or released from the nucleotide when the nucleotide is incorporated into the strand of nucleic acid complementary to the nucleic acid template.

13. A method according to claim 12 comprising determining nature and/or quantity of label cleaved or released from one or more nucleotides incorporated into the strand of nucleic acid complementary to the nucleic acid template.

14. A method according to claim 5 comprising performing a cycle of sets of steps wherein within each set of steps in the cycle three nucleotides are provided in a first step and one nucleotide is provided in a second step.

15. A method according to claim 14 comprising performing four cycles of sets of steps for said nucleic acid, wherein within each of the cycles the one nucleotide provided in all the second steps of all the sets of steps is the same, and wherein the one nucleotide provided in all the second steps of all the sets of steps in each cycle is different from the one nucleotide provided in all the second steps of all the sets of steps in the other three cycles.

16. A method according to claim 1, wherein a set of steps additionally comprises providing one or more blocked nucleotides that stop incorporation of nucleotides into the strand of nucleic acid complementary to the nucleic acid template.

17. A method according to claim 1, wherein a set of steps additionally comprises providing one or more non-incorporating inhibitor nucleotides which inhibit misincorporation of nucleotides into the strand of nucleic acid complementary to the nucleic acid template.

18. A method according to claim 1 wherein the nucleic acid template is a deoxyribonucleic acid (DNA), the nucleic acid polymerase is a DNA-dependent DNA polymerase and the nucleotides are deoxyribonucleotides or deoxyribonucleotide analogs.

19. A method according to claim 1 wherein the nucleic acid template is a deoxyribonucleic acid (DNA), the nucleic acid polymerase is a DNA-dependent ribonucleic acid (RNA) polymerase and the nucleotides are ribonucleotides or ribonucleotide analogs.

20. A method according to claim 1 wherein the nucleic acid template is a ribonucleic acid (RNA), the nucleic acid polymerase is a reverse transcriptase and the nucleotides are deoxyribonucleotides or deoxyribonucleotide analogs.

21. A method according to claim 1 wherein the nucleic acid template is provided in multiple copies.

22. A method according to claim 21 comprising providing multiple copies of the nucleic acid template by a nucleic acid amplification reaction.

23. A method according to claim 22 wherein the nucleic acid amplification reaction comprises rolling circle amplification.

24. A method according to claim 23 comprising:

providing a DNA molecule consisting of a stem portion and first and second loop portions, wherein the stem portion consists of a first strand and a second strand, wherein the first strand and second strand are equal in length, complementary and annealed together and comprise a region for which sequence and/or base composition information is desired, wherein the first loop portion joins the 3′

end of the first strand to the 5′

end of the second strand and the second loop portion joins the 3′

end of the second strand to the 5′

end of the first strand so the DNA molecule has no free 5′

or 3′

ends, wherein a loop portion comprise a primer binding site for rolling-circle amplification and a loop portion comprises a primer binding site for sequencing;

performing rolling circle amplification to provide multiple copies of the nucleic acid to serve as said nucleic acid template.

25. A method according to claim 1 wherein the nucleic acid template is attached to a solid support.

26. A method according to claim 25 wherein multiple different nucleic acid templates are attached to a solid support in an array.

27. A method according to claim 25 wherein the nucleic acid template is attached to the solid support via annealing to a primer that is attached to the solid support.

28. A method according to claim 1 comprising determining the sequence of a nucleic acid by analysis of determination of nature and/or quantity of nucleotides incorporated into the strand of nucleic acid complementary to the nucleic acid template.

31. A computer processor programmed to control a method of according to claim 1.

32. A computer-readable device carrying a program for a computer processor according to claim 31.

33. A computer processor programmed to provide sequence and/or base composition information for a nucleic acid from performance of a method according to claim 1.

34. A computer-readable device carrying a program for a computer processor according to claim 33.

35. A reagent kit suitable for performing a method according to claim 1, the reagent kit including one or more sets of premixed reagents in one or more reagent vessels, wherein each set of premixed reagents comprises

nucleotides taken from all four complementarity classes,

at least one vessel containing nucleotides taken from more than one, optionally two, three or four, complementarity classes, and the nucleotides in at least one of the complementarity classes, if incorporated into the strand of nucleic acid complementary to a nucleic acid template, allow further elongation of the strand of nucleic acid complementary to the nucleic acid template, and

wherein if nucleotides taken from all four complementarity classes are provided in a single vessel then the nucleotides in one, two or three of the of the complementarity classes, if incorporated into the strand of nucleic acid complementary to the nucleic acid template, prevent further elongation of the strand of nucleic acid complementary to the nucleic acid template.

36. An instrument for performing a method according to claim 1, comprising:

an imaging component able to detect an incorporated or released label,

a reaction chamber for holding one or more attached templates such that they are accessible to the imaging component at least once per set of steps,

a reagent distribution system for providing reagents to the reaction chamber.

37. An instrument according to claim 36 wherein the reaction chamber provides, and the imaging component is able to resolve, attached templates at a density of at least 100/cm², optionally at least 1000/cm², at least 10000/cm² or at least 100000/cm².

38. An instrument according to claim 35 wherein the imaging component employs a system or device selected from the group consisting of photomultiplier tubes, photodiodes, charge-coupled devices, CMOS imaging chips, near-field scanning microscopes, far-field confocal microscopes, wide-field epi-illumination microscopes and total internal reflection miscroscopes.

39. An instrument according to claim 35 wherein the imaging component detects fluorescent labels.

40. An instrument according to claim 39 wherein the imaging component detects laser-induced fluorescence.

41. An instrument according to claim 35 wherein the reaction chamber is a closed structure comprising a transparent surface, a lid, and ports for attaching the reaction chamber to the reagent distribution system, where the transparent surface holds template molecules on its inner surface and the imaging component is able to image through the transparent surface.

29. A nucleic acid sequencing-by-synthesis method characterized by incorporation of nucleotides in a step-wise manner, wherein a step allows for template-dependent incorporation of more than one different nucleotide.

30. A method according to claim 29 wherein a step allows for template-dependent incorporation of three different nucleotides selected from the group consisting of nucleotides complementary to Adenosine (A), Cytosine (C), Thymine (T) and Guanine (G), and a separate step allows for template-dependent incorporation of the remaining nucleotide of the group.

42. A DNA molecule consisting of a stem portion and first and second loop portions, wherein the stem portion consists of a first strand and a second strand, wherein the first strand and second strand are equal in length, complementary and annealed together, wherein the first loop portion joins the 3′

end of the first strand to the 5′

end of the second strand and the second loop portion joins the 3′

end of the second strand to the 5′

end of the first strand so the DNA molecule has no free 5′

or 3′

ends.

43. A DNA molecule according to claim 42 wherein a loop portion comprises a primer binding site for rolling-circle amplification.

44. A DNA molecule according to claim 42 wherein a loop portion comprises a primer binding site for sequencing.

45. An array of multiple different DNA molecules according to claim 42, attached to a solid support, optionally via annealing to primers attached to the solid support.

46. A method of making a DNA molecule according to claim 42 the method comprising:

providing a double-stranded DNA molecule consisting of

a first strand which has a 5′

end and a 3′

end, and

a second strand which has a 5′

end and a 3′

end; and

ligating a first linker to join the 3′

end of the first strand to the 5′

end of the second strand, and ligating a second linker to join the 3′

end of the second strand to the 5′

end of the first strand, wherein the linkers are hairpin structures.

47. A method of producing multiple copies of a DNA template, the method comprising performing rolling-circle amplification on a DNA molecule according to claim 43 to produce an elongated DNA molecule comprising multiple copies of the DNA template.

48. A method of producing multiple copies of multiple DNA templates, the method comprising performing rolling-circle amplification on multiple DNA molecules according to claim 43 to produce multiple elongated DNA molecules comprising multiple copies of the DNA templates.

49. A method according to claim 47 wherein a rolling circle amplification primer or the DNA molecules are attached to a solid support.

50. A method according to claim 47 further comprising condensing the elongated DNA molecules by annealing between complementary strands within the multiple copies of the DNA template within the elongated DNA molecules.

51. A method according to claim 50 wherein the elongated DNA molecules are condensed onto a solid support.

52. A method according to claim 47 further comprising sequencing multiple copies of the DNA template or DNA templates within the elongated DNA molecules.