Conversion of Target Specific Amplification to Universal Sequencing

• US 20080131937A1
• Filed: 06/15/2007
• Published: 06/05/2008
• Est. Priority Date: 06/22/2006
• Status: Abandoned Application

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Applera Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Schroeder, Benjamin G.
• Application Number:
  US11/764,115
• Publication Number:
  US 20080131937A1
• Time in Patent Office: Days
• US Class Current: 435/91.2
• CPC Class Codes: C12Q 1/6855 Ligating adaptors
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2521/313 Type II endonucleases, i.e. cutting outside recognition site
• CPC Class Codes: C12Q 2525/131 incorporating a restriction site
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor

Abstract

The present teachings provide methods, compositions, and kits for amplifying nucleic acids. The amplified nucleic acids can then be sequenced. The sequencing reaction for such amplified nucleic acids can provide additional sequence information, and less redundancy, as compared to conventional approaches. In some embodiments, a target polynucleotide is amplified with a primer to form an amplicon comprising a type IIs restriction enzyme recognition site. Following digestion with a type IIs restriction enzyme, and ligation of an adapter, a sequencing primer can be hybridized to the adapter, and a sequencing reaction performed. The sequencing reaction results in the omission of unwanted sequence information that was present in the amplification primer.

Claims

1. A method of performing a re-sequencing reaction comprising;

performing a polymerase chain reaction (PCR) with a forward primer and a reverse primer to form a PCR amplicon,

wherein the forward primer comprises a target-specific portion and a 5′

tail, wherein the 5′

tail of the forward primer comprises a type IIs restriction enzyme recognition site, and,

wherein the reverse primer comprises a target-specific portion and a 5′

tail, wherein the 5′

tail of the reverse primer comprises a type IIs restriction enzyme recognition site;

digesting the PCR amplicon with a type IIs restriction enzyme to form a digested amplicon with a first end and a second end;

ligating an adapter to the first end of the digested amplicon and ligating an adapter to the second end of the digested amplicon to form an adapter-ligated amplicon with a first end and a second end; and

,

sequencing the adapter-ligated amplicon.

2. The method according to claim 1 wherein the sequencing comprises

hybridizing a primer to the first end of the adapter-ligated amplicon;

and,

performing a chain termination reaction.

3. The method according to claim 1 wherein the sequencing comprises a cycled ligation.

4. The method according to claim 1 wherein the adapter ligated to the first end of the digested amplicon is different from the adapter ligated to the second end of the digested amplicon.

5. The method according to claim 1 wherein the type IIs restriction enzyme is Mme I or EcoP15I.

13. The method according to claim 1 wherein the type IIs restriction enzyme is Mme I or EcoP151.

22. The method according to claim 1 wherein the exonuclease is lambda exonuclease.

6. A method of forming an adapter-ligated amplicon comprising a first end that differs in sequence from a second end, said method comprising;

amplifying a short nucleic acid in a polymerase chain reaction (PCR) comprising a forward primer and a reverse primer to form a PCR amplicon,

wherein the forward primer comprises a target-specific portion and a 5′

tail, wherein the 5′

tail of the forward primer comprises a type IIs restriction enzyme recognition site, and,

wherein the reverse primer comprises a target-specific portion and a 5′

tail, wherein the 5′

tail of the reverse primer comprises a type IIs restriction enzyme recognition site;

digesting the PCR amplicon with a type IIs restriction enzyme to form a digested amplicon with a first end and a second end; and

,

ligating a first adapter to the first end of the digested amplicon and ligating a second adapter to the second end of the digested amplicon to form an adapter-ligated amplicon with a first end that differs in sequence from a second end.

7. The method according to claim 6 wherein the PCR amplicon is 18-100 nucleotides in length.

8. The method according to claim 6 wherein the type IIs restriction enzyme is Mme I or EcoP15I.

9. A method of increasing the percent of novel sequence information in a sequencing reaction of a digested PCR amplicon less than 100 bases in length comprising;

performing a polymerase chain reaction (PCR) with a forward primer and a reverse primer to form a PCR amplicon,

wherein the forward primer comprises a target-specific portion and a 5′

tail, wherein the 5′

tail of the forward primer comprises a type IIs restriction enzyme recognition site, and,

wherein the reverse primer comprises a target-specific portion and a 5′

tail, wherein the 5′

tail of the reverse primer comprises a type IIs restriction enzyme recognition site;

digesting the PCR amplicon with a type IIs restriction enzyme to form a digested PCR amplicon with a first end and a second end, wherein the digested PCR amplicon is less than one hundred bases in length;

ligating an adapter to the first end of the digested PCR amplicon and ligating an adapter to the second end of the digested PCR amplicon to form an adapter-ligated PCR amplicon with a first end and a second end;

sequencing the adapter-ligated amplicon; and

,

increasing the percent of novel sequence information in a sequencing reaction of the PCR amplicon as compared to sequencing a PCR amplicon that was amplified in a PCR not comprising primers with a type IIs restriction enzyme site.

10. The method according to claim 9 wherein the sequencing comprises

hybridizing a primer to the first end of the adapter-ligated amplicon;

and,

performing a chain termination reaction.

11. The method according to claim 9 wherein the sequencing comprises a cycled ligation.

12. The method according to claim 9 wherein the adapter ligated to the first end of the digested PCR amplicon is different from the adapter ligated to the second end of the digested PCR amplicon.

14. A method of sequencing a target polynucleotide comprising;

amplifying the target polynucleotide with at least one primer to form an amplicon comprising a type IIs restriction enzyme recognition site on at least one end;

digesting the PCR amplicon with a type IIs restriction enzyme to form a digested amplicon with at least one ligatable end;

ligating an adapter to the ligatable end of the digested amplicon to form an adapter-ligated amplicon with a known end;

hybridizing a sequencing primer to the known end; and

,

sequencing the adapter-ligated amplicon, wherein the first sequenced base is target polynucleotide information.

15. The method according to claim 14 wherein the sequencing comprises

hybridizing a primer to the first end of the adapter-ligated amplicon;

and,

performing a chain termination reaction.

16. The method according to claim 14 wherein the sequencing comprises a cycled ligation.

17. The method according to claim 14 wherein the type IIs restriction enzyme is Mme I or EcoP151.

18. A method of performing a sequencing reaction comprising;

performing a polymerase chain reaction (PCR) with a forward primer and a reverse primer to form a PCR amplicon,

wherein the forward primer comprises a target-specific portion and a 5′

tail, wherein the 5′

tail of the forward primer comprises an exonuclease-resistant moiety, and,

wherein the reverse primer comprises a target-specific portion and a 5′

tail, wherein the 5′

tail of the reverse primer comprises an exonuclease-resistant moiety;

digesting the PCR amplicon with an exonuclease to form a digested PCR amplicon with a first end and a second end;

ligating an adapter to the first end of the digested PCR amplicon and to the second end of the digested PCR amplicon to form an adapter-ligated amplicon with a first end and a second end; and

,

sequencing the adapter-ligated amplicon.

19. The method according to claim 18 wherein the sequencing comprises

hybridizing a primer to the first end of the adapter-ligated amplicon;

and,

performing a chain termination reaction.

20. The method according to claim 18 wherein the sequencing comprises a cycled ligation.

21. The method according to claim 18 wherein the adapter ligated to the first end of the digested amplicon comprises a sequence that is different from the adapter ligated to the second end of the digested amplicon.

23. A method of performing a sequencing reaction, wherein the first sequenced base is target polynucleotide information, said method comprising;

performing a primer-mediated amplification reaction to form an amplicon, wherein the primer comprises a target-specific portion and cleavage-related moiety;

digesting the amplicon with a cleavage agent directed to the cleavage-related moiety to form a digested amplicon with a cleaved end;

ligating an adapter to the cleaved end of the digested amplicon to form an adapter-ligated amplicon; and

,

sequencing the adapter-ligated amplicon, wherein the first sequenced base is target polynucleotide information.

24. The method according to claim 23 wherein the sequencing comprises

hybridizing a primer to the first end of the adapter-ligated amplicon;

and,

performing a chain termination reaction.

25. The method according to claim 23 wherein the sequencing comprises a cycled ligation.

26. The method according to claim 23 wherein the cleavage-related moiety is a type IIs restriction enzyme recognition site.

27. The method according to claim 26 wherein the type IIs restriction enzyme is Mme I or EcoP15I.

28. The method according to claim 23 wherein the cleavage-related moiety is an exonuclease-resistant moiety.

29. The method according to claim 28 wherein the exonuclease-resistant moiety is polyethylene glycol (PEG).

30. A kit comprising;

a forward primer comprising a target-specific portion and a 5′

tail, wherein the 5′

tail of the forward primer comprises a type IIs restriction enzyme recognition site;

a reverse primer comprising a target-specific portion and a 5′

tail, wherein the 5′

tail of the reverse primer comprises a type IIs restriction enzyme recognition site;

a first adapter comprising a first universal primer portion;

a second adapter comprising a second universal primer portion;

a ligase;

a polymerase; and

,

dNTPs.

31. The kit according to claim 30 further comprising nucleotide terminators.

32. The kit according to claim 30 further comprising a first sequencing primer and a second sequencing primer, wherein the first sequencing primer is encoded by the first universal primer portion of the first adapter and wherein the second sequencing primer is encoded by the second universal primer portion of the second adapter.

33. The kit according to claim 32 wherein the first sequencing primer, the second sequencing primer, or both the first sequencing primer and the second sequencing primer contain a fluorescent label.

34. The kit according to claim 30 further comprising sequencing ligation probes.

35. A composition comprising;

a digested PCR amplicon less than 50 nucleotides in length, the digested PCR amplicon comprising a first single-stranded end resulting from cleavage of a first type IIs restriction enzyme recognition site by a type IIs restriction enzyme that was introduced by a forward primer, and, a second single-stranded end resulting from cleavage of a type IIs restriction enzyme site by a type IIs restriction enzyme that was introduced by a reverse primer.

36. The composition according to claim 35 where the first single-stranded end is 1-4 bases in length, the second single-stranded end is 1-4 bases in length, or the first single-stranded end is 1-4 bases in length and the second single-stranded end are both 1-4 bases in length.

37. The composition according to claim 35 wherein the first single-stranded end, the second single-stranded end, or both the first single-stranded end and the second single-stranded end result from cleavage with Mme I or EcoP15I.

38. A kit comprising;

a forward primer comprising a target-specific portion and a 5′

tail, wherein the 5′

tail of the forward primer comprises a exonuclease-resistant moiety;

a reverse primer comprising a target-specific portion and a 5′

tail, wherein the 5′

tail of the reverse primer comprises an exonuclease-resistant moiety;

a first adapter comprising a first universal primer portion;

a second adapter comprising a second universal primer portion;

a ligase;

a polymerase; and

,

dNTPs.

39. The kit according to claim 38 further comprising nucleotide terminators.

40. The kit according to claim 38 further comprising a first sequencing primer and a second sequencing primer, wherein the first sequencing primer is encoded by the first universal primer portion of the first adapter and wherein the second sequencing primer is encoded by the second universal primer portion of the second adapter.

41. The kit according to claim 40 wherein the first sequencing primer, the second sequencing primer, or both the first sequencing primer and the second sequencing primer contain a fluorescent label.

42. The kit according to claim 38 further comprising sequencing ligation probes.

43. A composition comprising;

a digested PCR amplicon less than 50 nucleotides in length, the digested PCR amplicon comprising a first single-stranded end resulting from cleavage with a first exonuclease of a region upstream from a first exonuclease-resistant moiety that was introduced by a forward primer, and, a second single-stranded end resulting from cleavage with a second exonuclease of a region upstream from a second exonuclease-resistant moiety that was introduced by a reverse primer.

44. The composition according to claim 43 where the first single-stranded end is 1-4 bases in length, the second single-stranded end is 1-4 bases in length, or the first single-stranded end is 1-4 bases in length and the second single-stranded end are both 1-4 bases in length.

45. The composition according to claim 43 wherein the first exonuclease and the second exonuclease are the same exonuclease.

46. A kit comprising;

a forward primer comprising a target-specific portion and a 5′

tail, wherein the 5′

tail of the forward primer comprises cleavage-related moiety;

a reverse primer comprising a target-specific portion and a 5′

tail, wherein the 5′

tail of the reverse primer comprises a cleavage-related moiety;

a first adapter comprising a first universal primer portion;

a second adapter comprising a second universal primer portion;

a ligase;

a polymerase; and

,

dNTPs.

47. The kit according to claim 46 further comprising nucleotide terminators.

48. The kit according to claim 46 further comprising a first sequencing primer and a second sequencing primer, wherein the first sequencing primer is encoded by the first universal primer portion of the first adapter and wherein the second sequencing primer is encoded by the second universal primer portion of the second adapter.

49. The kit according to claim 48 wherein the first sequencing primer, the second sequencing primer, or both the first sequencing primer and the second sequencing primer contain a fluorescent label.

50. The kit according to claim 46 wherein the cleavage-related moiety is a type IIs restriction enzyme recognition site.

51. The kit according to claim 46 wherein the cleavage-related moiety is an exonuclease resistant moiety.

52. The kit according to claim 46 further comprising sequencing ligation probes.

53. A composition comprising;

a digested amplicon ligated to an adapter, and,

a sequencing primer hybridized to the adapter,

wherein the digested amplicon is less than 50 nucleotides in length and comprises a single-stranded end hybridized to the adapter, wherein the single-stranded end results from cleavage of a cleavage-related moiety introduced by an amplification primer.

54. The composition according to claim 53 wherein the cleavage-related moiety introduced by the amplification primer is a type IIs restriction enzyme recognition site.

55. The composition according to claim 53 wherein the cleavage-related moiety introduced by the amplification primer is an exonuclease resistant moiety.

56. The composition according to claim 53 wherein the amplification primer is a PCR primer, and wherein the digested amplicon is a digested PCR amplicon.

57. The composition according to claim 53 wherein the amplification primer is a rolling circle amplification primer, and wherein the digested amplicon is a digested rolling circle amplification reaction product.