Nucleic acid production and sequence analysis

Patent metadata

Field	Value
Publication number	US-9347093-B2
Application number	US-201213679538-A
Country	US
Kind code	B2
Filing date	Nov 16, 2012
Priority date	Nov 17, 2011
Publication date	May 24, 2016
Grant date	May 24, 2016

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.

First claim

What is claimed is:

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.

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.HsaDnmt 3 B, 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

Assignees

• Univ Vilnius

Inventors

• Klimasauskas Saulius
• Stasevskij Zdislav

Classifications

• C12Q1/6876

  Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes · CPC title

• C12Q1/6846

  Common amplification features · CPC title

• C12Q1/6853 Primary

  using modified primers or templates · CPC title

• C12P19/34

  Polynucleotides, e.g. nucleic acids, oligoribonucleotides · CPC title

• C12Q1/6869

  Methods for sequencing · CPC title