Method for monitoring the temperature of a biochemical reaction

• US 7,258,979 B2
• Filed: 11/19/2003
• Issued: 08/21/2007
• Est. Priority Date: 09/07/1998
• Status: Expired due to Fees

• Current Assignee: Secretary of State For Defence
• Original Assignee: Secretary of State For Defence
• Inventors: Brightwell, Gale, Lee, Martin A
• Primary Examiner(s): WHISENANT, ETHAN C
• Application Number:
  US10/715,407
• Publication Number:
  US 20040106144A1
• Time in Patent Office: 1,371 Days
• Field of Search: 435/6, 536/23.1, 536/24.3
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/6851 Quantitative amplification
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides
• CPC Class Codes: C12Q 2527/107 Temperature of melting, i.e. Tm
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: C12Q 2563/173 staining/intercalating agent, e.g. ethidium bromide
• CPC Class Codes: C12Q 2565/101 Interaction between at least two labels
• CPC Class Codes: C12Q 2565/1015 labels being on the same oligonucleotide
• CPC Class Codes: C12Q 2565/107 Alteration in the property of hybridised versus free label oligonucleotides

Abstract

A method of monitoring the temperature of a biochemical reaction such as an amplification reaction is described. The method comprises effecting the reaction in the presence of a fluorescently labeled temperature probe DNA sequence which comprises a double stranded region which denatures at a predetermined temperature, the fluorescent label of said temperature probe sequence being arranged so that a detectable signal occurs at the point at which denaturation of said region takes places; and monitoring fluorescence from said reaction mixture so as to determine when said predetermined temperature has been reached.

Claims

1. A method of monitoring the temperature of a biochemical reaction in a reaction mixture, said method comprising:

(i) designing a fluorescently labelled temperature probe DNA sequence so that it comprises a double stranded region which denatures at any desired predetermined temperature wherein the fluorescent label of said temperature probe sequence is arranged so that a detectable signal occurs at the point at which denaturation of the said region takes place;

(ii) effecting said reaction in the presence of said probe; and

(iii) monitoring fluorescence from said reaction mixture so as to determine when said predetermined temperature has been reached.

2. A method according to claim 1 wherein the temperature probe DNA sequence comprises a labelled double stranded DNA sequence.

3. A method according to claim 1 wherein the temperature probe DNA sequence comprises a single nucleic acid strand, end regions of which hybridize together so as to form a loop or “

hairpin”

structure.

4. A method according to claim 1 wherein the fluorescent label comprises an intercalating dye.

5. A method according to claim 1 wherein the fluorescent label used in the method employs fluorescence transfer (FRET) as the basis of the signal.

6. A method according to claim 5 wherein the temperature probe DNA sequence is provided with a reporter and a quencher molecule, arranged so that the hybridization of the denatured strands alters the spatial relationship between the quencher and reporter molecules.

7. A method according to claim 6 wherein the temperature probe sequence is a single stranded sequence, where the end portions hybridize together and wherein the reporter molecule is attached in the region of either the 5′

or the 3′

end of the sequence and the quencher molecule is attached at the opposite end.

8. A method according to claim 6 wherein the reporter and quencher molecules are located on different strands of a DNA temperature probe sequence such that on hybridization of the strands, they are brought into close proximity to each other.

9. A method according to claim 8 wherein FRET is established between an intercalating dye and a quencher molecule arranged on a strand of the temperature probe sequence such that is can absorb radiation from dye which is in close proximity on hybridization of the strands.

10. A method according to claim 6 wherein the temperature probe DNA sequence comprises a first DNA strand having a reporter molecule thereon, a second DNA strand having a quencher molecule thereon, said first and second DNA strands being designed to hybridize to a third DNA strand such that the reporter and quencher molecules are brought into close proximity with each other.

11. A method according to claim 1 wherein the pre-determined temperature at which the DNA sequence denatures is determined by the length of the temperature probe sequence.

12. A method according to claim 1 wherein the pre-determined temperature at which the DNA sequence denatures is determined by the GC content of the sequence.

13. A method according to claim 1 wherein the biochemical reaction is an amplification reaction.

14. A method according to claim 13 wherein the amplification reaction is a polymerase chain reaction (PCR).