System for multi color real time pcr

• US 20060269922A1
• Filed: 04/01/2004
• Published: 11/30/2006
• Est. Priority Date: 04/04/2003
• Status: Active Grant

• Current Assignee: Roche Molecular Systems (Roche Holding AG)
• Original Assignee: Roche Diagnostics Operations Incorporated (Roche Holding AG)
• Inventors: Mueller, Christoph, Bechler, Ingrid, Heindl, Dieter, Josel, Hans-Peter, Sagner, Gregor, Gutekunst, Martin, Bolte, Jochen, Sebl, Rudolf
• Granted Patent: US 8,137,616 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6851 Quantitative amplification
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2561/113 Real time assay
• CPC Class Codes: C12Q 2565/101 Interaction between at least two labels
• CPC Class Codes: G01N 2021/6421 Measuring at two or more wavelengths

Abstract

The invention is directed to a system for performing multi-color real time PCT, comprising a flexible real time PCT instrument and a specific composition or reaction mixture for performing multiplex PCR: In particular, the present invention is directed to a composition or reaction mixtuer which comprises at least 3, preferably 4-5 and most preferably exactly 4 pairs of FRET hybridization probes. Each pair of said hybridization probes consists of a FRET donor probe carrying a FRET donor moiety and a FRET acceptor probe carrying a FRET acceptor moiety having an emission maximum between 550 and 710 nm.

Claims

1. A composition or reaction mixture for performing multi-color real time PCR comprising at least 3 pairs, of FRET hybridization probes, each pair of hybridization probes consisting of a FRET donor probe carrying a FRET donor moiety and a FRET acceptor probe carrying a FRET acceptor moiety having an emission maximum between 550 and 710 nm.

2. A composition or reaction mixture according to claim 1, wherein at least 3 FRET donor moieties are identical.

3. A composition or reaction mixture according to claim 1, wherein all FRET donor moieties are identical.

4. A composition or reaction mixture according to claim 1, wherein at least 3 FRET donor moieties are Fluorescein.

5. A composition or reaction mixture according to claim 1, wherein all FRET donor moieties are Fluorescein.

6. A composition or reaction mixture according to claims 1, wherein at least one additional FRET donor moiety is selected from a group consisting of Atto425 and WI343.

7. A composition or reaction mixture according to claims 1, wherein one FRET acceptor moiety is selected from a group consisting of LC-Red 705, Cy5.5, and JA286.

8. A composition or reaction mixture according to claims 1, wherein at least one FRET acceptor moiety is selected from a group consisting of Cy5, LC-Red 640, and LC-Red 610.

9. A composition or reaction mixture according to claims 1, wherein one FRET acceptor moiety is selected from a group consisting of Rh6G and TAMRA.

10. A system for performing multi-color real time PCR, comprising

a real time PCR instrument, and

a composition or reaction mixture according to claims 1.

11. A system according to claim 10, characterized in that said real time PCR instrument comprises

at least 1 light source

at least 4 fluorescent detector entities, each of said entities having central detection wavelengths which are distinct from each other by at least 25-30 nm.

wherein said detector entities are capable of

simultaneously detecting maximum fluorescence emission of at least 3, differently labeled FRET Hybridization Probe pairs,

simultaneously detecting maximum fluorescence emission of at least 2 differently labeled TaqMan hybridization probes, and

detecting maximum fluorescence emission of SybrGreenI

means for heating and cooling and

multiple reaction vessels for containing a reaction mixture.

12. A system according to claim 11, wherein said real time PCR instrument comprises exactly one light source.

13. A method for amplifying and detecting multiple target DNA sequences comprising:

a) providing a composition or reaction mixture according to claim 1,

b) subjecting said reaction mixture to a thermocycling protocol such that amplification of said multiple target sequences can take place, and

c) monitoring hybridization of each of said pairs of FRET hybridization probes at least once after a plurality of amplification cycles.

14. A method according to claim 13, wherein hybridization is monitored at least once in a temperature dependent manner.

15. A real time PCR instrument comprising

at least 1 light source,

at least 5 fluorescent detector entities, each of said entities having central detection wavelengths which are distinct from each other by at least 25-30 nm,

characterized in that said detector entities are capable of

simultaneously detecting maximum fluorescence emission of at least 3 differently labeled FRET Hybridization Probe pairs,

simultaneously detecting maximum fluorescence emission of at least 2 differently labeled TaqMan hybridization probes, and

detecting maximum fluorescence emission of SybrGreenI

means for heating and cooling, and

multiple reaction vessels for containing a reaction mixture.

16. A real time PCR instrument according to claim 15 comprising one light source.

17. An instrument according to claim 15, wherein said central detection wavelengths are selected from a group of range of wavelengths, said group consisting of 520-540 nm, 545-565 nm, 570-590 nm, 600-620 nm, 630-650 nm, 660-680 nm, and 700-720 nm.