Method of generating size standard nucleic acids

• US 20050095610A1
• Filed: 04/07/2004
• Published: 05/05/2005
• Est. Priority Date: 04/11/2003
• Status: Abandoned Application

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Inventors: Chen, Shiaw-Min, Chang, Chu-an, Spurgeon, Sandra L., Kuo, Sophia S.
• Application Number:
  US10/819,657
• Publication Number:
  US 20050095610A1
• Time in Patent Office: Days
• US Class Current: 435/6.12
• 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: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 2525/151 repeat or repeated sequences, e.g. VNTR, microsatellite, concatemer
• CPC Class Codes: C12Q 2525/204 specific length of the oligonucleotides
• CPC Class Codes: C12Q 2533/101 Primer extension

Abstract

Methods for generating nucleic acid size standards are disclosed. The methods comprise providing a template polynucleotide which comprises periodic sequences of from about 5 to about 50 contiguous nucleotides containing not more than three types of nucleotides and wherein adjacent periodic sequences are separated by a terminator complement nucleotide that differs from the not more than three types of nucleotides, and performing a primer extension reaction on the template polynucleotide in the presence of nucleoside triphosphate molecules and a 3′ terminating nucleoside triphosphate which is complementary to the terminator complement nucleotide.

Claims

1. A method for generating nucleic acid size standards, the method comprising:

providing a template polynucleotide which comprises periodic sequences of from about 5 to about 50 contiguous nucleotides containing not more than three different types of nucleotides and wherein adjacent periodic sequences are separated by a terminator complement type of nucleotide different from each of the not more than three different types of nucleotides; and

performing a primer extension reaction on the template polynucleotide in the presence of a primer which is sufficiently complementary to the template polynucleotide to hybridize therewith, a nucleic acid polymerase, nucleoside triphosphate molecules suitable for a polymerase extension of the primer on the template polynucleotide and a 3′

terminating nucleoside triphosphate which is complementary to the terminator complement type of nucleotide.

2. A method according to claim 1 wherein the template polynucleotide is an artificial sequence.

3. A method according to claim 1 wherein the periodic sequences are substantially of the same length.

4. A method according to claim 3 wherein the periodic sequences are about 25 nucleotides in length.

5. A method according to claim 1 wherein the periodic sequences contain from about 6 to about 20 contiguous nucleotides.

6. A method according to claim 1 wherein the template polynucleotide is at least about 500 nucleotides in length and the method generates size standards which are from about 25 contiguous nucleotides to at least about 500 contiguous nucleotides in length.

7. A method according to claim 6 wherein about 20 size standard fragments are generated.

8. A method according to claim 6 wherein the template polynucleotide is about 1000 nucleotides in length and the method generates size standards which are from about 25 contiguous nucleotides to about 1000 contiguous nucleotides in length.

9. A method according to claim 8 wherein about 40 size standard fragments are generated.

10. A method according to claim 1 wherein the 3′

terminating nucleoside triphosphate is a dideoxynucleoside triphosphate or a 3′

amino nucleoside triphosphate.

11. A method according to claim 1 wherein the 3′

terminating nucleoside triphosphate further comprises a covalently attached label.

12. A method according to claim 11 wherein the label is a fluorophore, a chromophore, a biotin, a hapten, a radioisotope, a chemiluminescent moiety, or a spin label.

13. A method according to claim 12 wherein the label is a fluorophore selected from the group consisting of VIC, FAM, ROX, LIZ and TAMRA.

14. A method according to claim 12 wherein the label is a radioisotope selected from the group consisting of ³H, ¹⁴C, ³²p, and ³³P.

15. A method according to claim 1 wherein some of the periodic sequences contain one or more landmark nucleotides.

16. A method according to claim 15 wherein the one or more landmark nucleotides is a triplet of nucleotides comprising the identical terminator complement type of nucleotide.

17. A method according to claim 1 wherein the template polynucleotide contains restriction sites suitable for cloning the template.

18. A method according to claim 1 wherein the periodic sequences are random sequences of the not more than three different types of nucleotides.

19. A method according to claim 18 wherein the not more than three different types of nucleotides comprise not more than two different types of nucleotides.

20. A method according to claim 19 wherein the not more than two different types of nucleotides comprise not more than one type of nucleotide.

21. A method according to claim 1 wherein the primer further comprises a covalently attached label.

22. A method according to claim 21 wherein the label is a fluorophore, a chromophore, a biotin, a hapten, a radioisotope, a chemiluminescent moiety, or a spin label.

23. A method according to claim 22 wherein the label is a fluorophore selected from the group consisting of VIC, FAM, ROX, LIZ and TAMRA.

24. A method according to claim 23 wherein the label is a radioisotope selected from the group consisting of ³H, ¹⁴C, ³²p, and ³³P.

25. A method for generating nucleic acid size standards, the method comprising:

combining in a mixture, a template polynucleotide having a 5′

portion and a 3′

template portion, a primer which is sufficiently complementary to the 3′

portion of the polynucleotide to hybridize therewith, a nucleic acid polymerase, nucleoside triphosphate molecules suitable for a polymerase extension of the primer on the template polynucleotide and a 3′

terminating nucleoside triphosphate; and

maintaining the mixture under conditions suitable for a primer extension reaction, wherein the template polynucleotide comprises periodic sequences of from about 5 to about 50 contiguous nucleotides none of which are complements to the 3′

terminating nucleoside triphosphate and wherein adjacent periodic sequences are separated by the complementary nucleotide of the 3′

terminating nucleoside triphosphate.

26. A method according to claim 25 wherein the template polynucleotide is an artificial sequence.

27. A method according to claim 25 wherein the periodic sequences are substantially of the same length.

28. A method according to claim 27 wherein the periodic sequences are about 25 nucleotides in length.

29. A method according to claim 25 wherein the periodic sequences contain from about 6 to about 20 contiguous nucleotides.

30. A method according to claim 25 wherein the template polynucleotide is at least about 500 nucleotides in length and the method generates size standards which are from about 25 contiguous nucleotides to at least about 500 contiguous nucleotides in length.

31. A method according to claim 30 wherein about 20 size standard fragments are generated.

32. A method according to claim 30 wherein the template polynucleotide is about 1000 nucleotides in length and the method generates size standards which are from about 25 contiguous nucleotides to about 1000 contiguous nucleotides in length.

33. A method according to claim 32 wherein about 40 size standard fragments are generated.

34. A method according to claim 25 wherein the 3′

terminating nucleoside triphosphate is a dideoxynucleoside triphosphate or a 3′

amino nucleoside triphosphate.

35. A method according to claim 25 wherein the 3′

terminating nucleoside triphosphate further comprises a covalently attached label.

36. A method according to claim 35 wherein the label is a fluorophore, a chromophore, a biotin, a hapten, a radioisotope, a chemiluminescent moiety, or a spin label.

37. A method according to claim 36 wherein the label is a fluorophore selected from the group consisting of VIC, FAM, ROX, LIZ and TAMRA.

38. A method according to claim 36 wherein the label is a radioisotope selected from the group consisting of ³H, ¹⁴C, ³²p, and ³³P.

39. A method according to claim 25 wherein some of the periodic sequences contain one or more landmark nucleotides.

40. A method according to claim 39 wherein the one or more landmark nucleotides is a triplet of nucleotides comprising the identical type of terminator nucleotide.

41. A method according to claim 25 wherein the template polynucleotide contains restriction sites suitable for cloning the template.

42. A method according to claim 25 wherein the periodic sequences are random sequences of not more than three types of nucleotides.

43. A method according to claim 42 wherein the not more than three different types of nucleotides comprise not more than two different types of nucleotides.

44. A method according to claim 43 wherein the not more than two different types of nucleotides comprise not more than one type of nucleotide.

45. A method according to claim 25 wherein the primer further comprises a covalently attached label.

46. A method according to claim 45 wherein the label is a fluorophore, a chromophore, a biotin, a hapten, a radioisotope, a chemiluminescent moiety, or a spin label.

47. A method according to claim 46 wherein the label is a fluorophore selected from the group consisting of VIC, FAM, ROX, LIZ and TAMRA.

48. A method according to claim 46 wherein the label is a radioisotope selected from the group consisting of ³H, ¹⁴C, ³²p, and ³³P.