KIT AND METHOD

• US 20130071846A1
• Filed: 05/13/2011
• Published: 03/21/2013
• Est. Priority Date: 05/14/2010
• Status: Active Grant

• Current Assignee: Biovica International AB
• Original Assignee: Per Stalhandske
• Inventors: Stlhandske, Per, Lennerstrand, Johan
• Granted Patent: US 9,429,518 B2
• Time in Patent Office: Days
• US Class Current: 435/6.11
• CPC Class Codes: C12N 9/1252 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
• CPC Class Codes: C12Q 1/485 involving kinase
• CPC Class Codes: C12Y 207/07007 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
• CPC Class Codes: G01N 21/6428 Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" in vivo A61B5/00; immunoassay G01N33/53
• CPC Class Codes: G01N 2333/91235 with a phosphate group as acceptor (2.7.4)
• CPC Class Codes: G01N 2333/9126 DNA-directed DNA polymerase (2.7.7.7)

Abstract

The present invention relates to a kit comprising a DNA-dependant DNA polymerase and at least one natural deoxynucleoside and to a kit comprising a DNA-dependent DNA polymerase and a detection system comprising a DNA template molecule, a DNA primer molecule, and a fluorescent moiety capable of being displaced from,or bound to, dsDNA synthesized by said DNA-dependent polymerase. It further relates to A method for measuring deoxynucleoside kinase activity in a sample characterized by; (i) contacting the sample, in a container, with a reaction mix comprising a DNA-dependent DNA polymerase, at least one deoxynucleoside and a detection system comprising a DNA template molecule, a DNA primer molecule, and a fluorescent moiety capable of being incorporated in, displaced from,or bound to dsDNA synthesized by said DNA dependent polymerase; (ii) incubating said container; (iii) measuring the signal from the fluorescent moiety;and correlating the signal from the fluorescent moietyto the deoxynucleoside kinase activity in the sample.

Claims

1. A kit comprising a DNA-dependant DNA polymerase and at least one natural deoxynucleoside.

3. A kit according to claim 1 or claim 2, further comprising at least a phosphotransferase.

4. A kit according to claim 1, further comprising at least one natural or modified deoxynucleoside.

5. A kit according to claim 4, wherein the at least one natural deoxynucleoside is chosen from deoxythymidine and/or deoxycytidine or a mix thereof.

6. A kit according to claim 4, wherein the at least one modified deoxynucleoside is chosen from modified thymidine and modified cytidine, such as BrdU and/or IdU, or a mix thereof.

2. A kit comprising a DNA-dependent DNA polymerase and a detection system comprising a DNA template molecule, a DNA primer molecule, and a fluorescent moiety capable of being displaced from, or bound to, dsDNA synthesized by said DNA-dependent polymerase.

7. A kit according to claim 2, wherein the fluorescent moiety is coupled to a single stranded DNA probe.

8. A kit according to claim 7, wherein the fluorescent moiety and a quenching moiety are coupled to a single stranded DNA probe.

9. A kit according to claim 2, wherein the fluorescent moiety is a double stranded DNA surface binding or intercalating molecule, such as PicoGreen, Eva dye or SYBR GreenI.

10. A kit according to claim 7, wherein the fluorescent moiety bound to the single stranded DNA probe is quenched when said probe is in its intact form.

11. A kit according to claim 7, wherein the DNA-dependant DNA polymerase has 5′

to 3′

exonuclease activity, such as Taq DNA polymerase or Tth polymerase.

12. A kit according to claim 7, wherein the fluorescent moiety bound to the single stranded DNA probe attains maximum fluorescence when in its intact form and hybridized to its target template.

13. A method for measuring deoxynucleoside kinase activity in a sample characterized by;

a. contacting the sample, in a container, with a reaction mix comprising a DNA-dependent DNA polymerase, at least one deoxynucleoside and a detection system comprising a DNA template molecule, a DNA primer molecule, and a fluorescent moiety capable of being incorporated in, displaced from, or bound to dsDNA synthesized by said DNA dependent polymerase;

b. incubating said container;

c. measuring the signal from the fluorescent moiety;

d. correlating the signal from the fluorescent moiety to the deoxynucleoside kinase activity in the sample.

14. A method according to claim 13, wherein the at least one deoxynucleoside is a natural or a modified deoxynucleoside or a mix thereof.

15. A method according to any of claim 13 or 14, wherein the at least one natural deoxynucleoside is chosen from deoxythymidine and/or deoxycytidine or mix a thereof.

16. A method according to any of claim 13 or 14, wherein the at least one modified deoxynucleoside is chosen from modified thymidine and modified cytidine, such as BrdU and/or IdU, or a mix thereof.

17. A method according to claim 13, wherein the fluorescent moiety is coupled to a single stranded DNA probe.

18. A method according to claim 13, wherein the fluorescent moiety and a quenching moiety are coupled to a single stranded DNA probe.

19. A method according to claim 13, wherein the fluorescent moiety is a double stranded DNA surface binding or intercalating molecule, such as PicoGreen, Eva dye or SYBR GreenI.

20. A method according to claim 17, wherein the fluorescent moiety bound to the single stranded DNA probe is quenched when said probe is in its intact form.

21. A method according to claim 17, wherein the DNA-dependant DNA polymerase has 5′

to 3′

exonuclease activity, such as Taq DNA polymerase or Tth polymerase.

22. A method according to claim 17, wherein the fluorescent moiety bound to the single stranded DNA probe attains maximum fluorescence when in its intact form and hybridized to its target template.