Method of monitoring the temperature of a biochemical reaction

• US 6,730,478 B1
• Filed: 03/06/2001
• Issued: 05/04/2004
• Est. Priority Date: 09/07/1998
• Status: Expired due to Fees

• Current Assignee: Secretary of State For Defence
• Original Assignee: The secretary of state for defence in her Britannic Majesty's Government of the United Kingdom of Great Britain And Northern Ireland
• Inventors: Brightwell, Gale, Lee, Martin A
• Primary Examiner(s): WHISENANT, ETHAN C
• Application Number:
  US09/786,521
• Time in Patent Office: 1,155 Days
• Field of Search: 435/6, 435/91.2, 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 for monitoring the temperature of a biochemical reaction is disclosed. The method effects the reaction in the presence of a fluorescently labeled DNA probe which comprises a double stranded region which dentures at a predetermined temperature. The fluorescent label is arranged so that a detectable signal is generated upon denaturation of the probe.

Claims

1. A method of monitoring the temperature of a biochemical reaction in a reaction mixture, said method comprising effecting the reaction in the presence of a fluorescently labelled 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 place;

and monitoring fluorescence from said reaction mixture so as to determine when said predetermined temperature has been reached, wherein the temperature probe comprises a single nucleic acid strand, end regions of which hybridise together so as to form a loop or hairpin structure and wherein the fluorescent label comprises an intercalating dye.

5. A method according to claim 1, 2, 3 or 4 wherein the biochemical reaction is an amplification reaction.

6. A method according to claim 5 wherein the amplification reaction is a polymerase chain reaction.

7. A method according to claim 6 wherein the length of the temperature probe is similar to that of an amplicon of the polymerase chain reaction.

2. A method of monitoring the temperature of a biochemical reaction in a reaction mixture, said method comprising effecting the reaction in the presence of a fluorescently labelled 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 place;

and monitoring fluorescence from said reaction mixture so as to determine when said predetermined temperature hasp been reached, wherein the fluorescent label utilises fluorescence transfer (FRET) as the basis of the signal and wherein the temperature probe DNA sequence is provided with a reporter and a quencher molecule, the reporter and quencher molecules being located on different strands of a DNA temperature probe sequence, and arranged such that on hybridisation of the strands, they are brought into close proximity to each other.

3. A method according to claim 2 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 hybridisation of the strands.

4. A method of monitoring the temperature of a biochemical reaction in a reaction mixture, said method comprising effecting the reaction in the presence of a fluorescently labelled 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 place;

and monitoring fluorescence from said reaction mixture so as to determine when said predetermined temperature has been reached, wherein the fluorescent label utilises fluorescence transfer (FRET) as the basis of the signal and 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 hybridise to a third DNA stand such that the reporter and quencher molecules are brought into close proximity with each other.