Kit and method

• US 9,429,518 B2
• Filed: 05/13/2011
• Issued: 08/30/2016
• Est. Priority Date: 05/14/2010
• Status: Active Grant

• Current Assignee: Biovica International AB
• Original Assignee: cSens AB (Biovica International AB)
• Inventors: Stlhandske, Per, Lennerstrand, Johan
• Primary Examiner(s): Switzer, Juliet
• Assistant Examiner(s): Dauner, Joseph G
• Application Number:
  US13/697,279
• Publication Number:
  US 20130071846A1
• Time in Patent Office: 1,936 Days
• US Class Current: 1/1
• 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 moiety to the deoxynucleoside kinase activity in the sample.

Claims

1. A homogenous method for real-time determination of deoxynucleoside kinase activity in a sample consisting of:

a) contacting the sample, in a container, with a reaction mix comprising a DNA-dependent DNA polymerase, at least one natural deoxynucleoside, at least one of cytidylate kinase and/or dTMP kinase, nucleoside-diphosphate kinase, 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 DNA polymerase;

b) closing and incubating said container;

c) measuring the signal from the fluorescent moiety during primer extension; and

d) correlating the signal from the fluorescent moiety to the deoxynucleoside kinase activity in the sample, wherein the method does not include any temperature cycling shifts.

2. A method according to claim 1, wherein the at least one natural deoxynucleoside is chosen from deoxythymidine and/or deoxycytidine or a mix thereof.

3. A method according to claim 1, wherein the fluorescent moiety is coupled to a single stranded DNA probe.

4. A method according to claim 3, wherein the fluorescent moiety bound to the single stranded DNA probe is quenched when said probe is in its intact form.

5. A method according to claim 3, wherein the DNA-dependent DNA polymerase has 5′

to 3′

exonuclease activity.

6. The method of claim 5, wherein the DNA-dependent DNA polymerase comprises Taq DNA polymerase or Tth polymerase.

7. The method of claim 5, wherein the DNA-dependent DNA polymerase comprises Taq DNA polymerase.

8. A method according to claim 3, wherein the fluorescent moiety bound to the single stranded DNA probe attains maximum fluorescence when the single stranded DNA probe is in its intact form and hybridized to its target template.

9. A method according to claim 1, wherein the fluorescent moiety and a quenching moiety are coupled to a single stranded DNA probe.

10. A method according to claim 1, wherein the fluorescent moiety is a double stranded DNA surface binding or intercalating molecule.

11. A method according to claim 1, wherein the temperature throughout steps a-d is kept at a value between 35°

and 42°

C.