Nucleic acid production and sequence analysis

• US 9,347,093 B2
• Filed: 11/16/2012
• Issued: 05/24/2016
• Est. Priority Date: 11/17/2011
• Status: Active Grant

• Current Assignee: Vilnius University
• Original Assignee: Vilnius University
• Inventors: Klimasauskas, Saulius, Stasevskij, Zdislav
• Primary Examiner(s): Mummert, Stephanie K
• Assistant Examiner(s): Priest, Aaron
• Application Number:
  US13/679,538
• Publication Number:
  US 20130130249A1
• Time in Patent Office: 1,285 Days
• Field of Search: None
• US Class Current: 1/1
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/6846 Common amplification features
• CPC Class Codes: C12Q 1/6853 using modified primers or templates
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 2521/125 Methyl transferase, i.e. methylase
• CPC Class Codes: C12Q 2525/117 incorporating modified base
• CPC Class Codes: C12Q 2525/155 incorporating/generating a new priming site
• CPC Class Codes: C12Q 2537/1373 Displacement by a nucleic acid

Abstract

A method for producing a nucleic acid molecule from a template nucleic acid sequence and a linking unit attached to a primer, which method comprises a step of contacting the template nucleic acid sequence with a nucleic acid polymerase under conditions which allow the nucleic acid polymerase to produce the nucleic acid molecule from the primer based on the template nucleic acid sequence, wherein the linking unit is attached to a target site in the template nucleic acid sequence with a covalent linkage.

Claims

1. A method for producing a nucleic acid molecule from a template nucleic acid sequence comprising a linking unit attached to a primer, which method comprises a step of contacting the template nucleic acid sequence with a nucleic acid polymerase under conditions which allow the nucleic acid polymerase to produce the nucleic acid molecule from the primer based on the template nucleic acid sequence, wherein the linking unit is attached with a covalent linkage to a target site within the template nucleic acid sequence and not to a terminal nucleotide of the template nucleic acid sequence such that priming of nucleic acid synthesis occurs at or within less than 5 bases of the target site.

2. The method according to claim 1 comprising a step of forming the covalent linkage using a transferase enzyme.

3. The method according to claim 1 wherein the primer comprises four nucleotides or less which are complementary to the template nucleic acid at the start point of production of the nucleic acid molecule by the nucleic acid polymerase.

4. The method according to claim 1 wherein the primer is covalently linked to the linking unit, or wherein the primer is non-covalently bound to the linking unit.

5. The method according to claim 4 wherein the linking unit comprises a nucleotide strand to which the primer is base paired.

6. The method according to claim 1 further comprising a step of forming the covalent linkage between the template nucleic acid sequence and the linking unit by contacting the target site derivatized with a first reactive group, with a second reactive group attached to the linking unit, under conditions that allow the first reactive group to react with the second reactive group to form the covalent linkage.

7. The method according to claim 6 further comprising a step of derivatizing the target site with a first reactive group.

8. The method according to claim 7 wherein the step of derivatizing the target site comprises contacting the template nucleic acid sequence with a compound and an enzyme, wherein the compound comprises the first reactive group, and wherein the enzyme is capable of transferring the first reactive group or a part of the compound comprising the first reactive group onto the target site.

9. The method according to claim 8 wherein when the part of the compound comprising the first reactive group is transferred onto the target site, the method further comprises a step of uncovering the first reactive group so that the first reactive group is available for reaction with the second reactive group.

10. The method according to claim 1, further comprising a step of determining the sequence of the produced nucleic acid.

11. The method according to claim 1, further comprising a step of amplifying the produced nucleic acid.

12. The method according to claim 11, further comprising a step of digesting the produced nucleic acid with a restriction enzyme.

13. The method according to claim 1 wherein the template nucleic acid sequence is DNA.

30. The method according to claim 7 wherein the step of derivatizing the target site with a first reactive group is carried out ex vivo.

14. A method for determining the presence or availability of a target site comprising a nucleotide within a template nucleic acid sequence, which method comprises:

(a) contacting the template nucleic acid sequence with a compound comprising a first reactive group and an enzyme, wherein the enzyme is capable of transferring the first reactive group, or a part of the compound comprising the first reactive group, onto the nucleotide of the target site;

(b) contacting the nucleic acid sequence with a second reactive group attached to a linking unit, optionally wherein the linking unit is attached to a primer, under conditions that allow the first reactive group to react with the second reactive group to form a covalent linkage;

(c) optionally, where the primer is not attached to the linking unit in step (b), binding a primer to the linking unit;

(d) contacting the nucleic acid sequence and the primer with a nucleic acid polymerase under conditions which allow the nucleic acid polymerase to produce a nucleic acid molecule from the primer based on the template nucleic acid sequence such that priming of nucleic acid synthesis occurs at or within less than 5 bases of the target site;

(e) detecting the presence or absence of the produced nucleic acid molecule so as to determine the presence or availability of the target site, wherein the covalent linkage formed in step (b) does not attach the linking unit to the template nucleic acid sequence via a terminal nucleotide of the template nucleic acid sequence, and wherein the enzyme is a DNA methyltransferase which is capable of using the compound as a co-factor.

15. The method according to claim 14 further comprising sequencing the produced nucleic acid molecule.

16. The method according to claim 14 further comprising a step of PCR amplification of the produced nucleic acid molecule.

17. The method according to claim 14, wherein when in step (a) the part of the compound comprising the first reactive group is transferred onto the nucleotide, the method further comprises a step prior to step (b) of uncovering the first reactive group.

18. The method according to claim 14 wherein the template nucleic acid sequence is an oligonucleotide.

19. The method according to claim 18 further comprising a step of forming the oligonucleotide by mechanical, enzymatic, or chemical digestion of DNA.

20. The method according to claim 19 wherein the step of mechanical digestion is DNA shearing.

21. The method according to claim 14 wherein the covalent linkage is selected from amide, thioureas, imidate, imine, thioether, disulfide, cyclic ester, hydrazone, oxime, thiaxolidine, 1,2,3-triazole, cyclohexene, arylalkyne, biaryl, and diyne.

22. The method according to claim 14 wherein the first reactive group or the second reactive group is an amine, a thiol, a 1,2,-diol, a hydrazine, a hydroxylamine, a 1,2-aminothiol, an azide, a diene, a terminal alkyne, an arylhalide or a terminal silylalkyne, an N-hydroxysuccinimidyl ester, a thioester, an isothiocyanate, an imidoester, an aldehyde, a ketone, a maleimide, a haloacetamide, an aziridine, an arylboronic acid, an alkyne, a phosphane ester, a dienophile, or a terminal haloalkyne.

23. The method according to claim 14 wherein the first reactive group is a terminal alkyne, an amine, a hydroxylamine, a thiol, a maleimide, an alkyne, a hydrazide, an azide, or a carbodiimide.

24. The method according to claim 14 wherein the DNA methyltransferase is M.Sssl, M.MpeORF4940P, M.Hhal, M.Hpall, M.HsaDnmt1, M.HsaDnmt3A, M.HsaDnmt3B, M.MmuDnmt1, M.MmuDnmt3A, M.MmuDnmt3B, M.Haelll, M.CviJl, M2.Eco31I., M.EcoRII, M.EcoDcm, M.Mval, M.BstNl, M.Taql, M.BseCl, M.Ecodam, T4Dam, M.Rsrl or M.EcoRl.

25. The method according to claim 14, wherein the target site comprises an unmethylated cytosine, an unmethylated adenine, or a 5-hydroxymethylcytosine.

26. The method according to claim 14 wherein the compound is an S adenosyl-L-methionine analog.

27. The method according to claim 14 wherein the linking unit comprises a nucleotide strand to which the primer is base paired in step (c).

28. The method according to claim 14 wherein the primer and/or the linking unit further comprise an affinity binding group.

29. The method according to claim 28 further comprising a step of enriching the produced nucleic acid molecules using affinity binding.

31. The method according to claim 14 wherein step (a) and optionally also step (b) is carried out ex vivo.

32. The method according to claim 14 wherein the compound is a thiol or a selenol.