System For Multi Color Real Time PCR

Abstract

The invention is directed to a system for performing multi-color real time PCR, comprising a flexible real time PCR instrument and a specific composition or reaction mixture for performing multiplex PCR: In particular, the present invention is directed to a composition or reaction mixture 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.

1 - 17 . (canceled) 
     
     
         18 . A real time PCR instrument comprising:
 One optical part for detecting fluorescence emission from a plurality of reaction vessels, the one optical part comprising:
 an excitation unit comprising:
 at least 1 light source capable of emitting light toward the reaction vessels containing fluorescent compounds, 
 a lightpipe being arranged for receiving light from the reaction vessel and capable of distributing homogeneously said light into optical fiber bundles, wherein the light does not pass through a wavelength excluding device prior to being distributed into said optical fiber bundles, 
 
 a detection unit comprising at least 5 separate fluorescent detector entities, each of said detector entities having a central detection wavelength, said wavelengths being distinct from each other by at least 25 nm, 
 a plurality of at least 5 optical fiber bundles, each of said bundle being arranged for-receiving homogeneously distributed light from the lightpipe, and transmitting said light to said fluorescent detector entities, and 
   means for heating and cooling,   wherein the excitation and detection units are located in separate housings inside the one optical part enabling highly precise positioning of the excitation unit towards each of the reaction vessels without moving the detection unit.   
     
     
         19 . A real time PCR instrument according to  claim 18 , comprising one light source. 
     
     
         20 . An instrument according to  claim 18 , 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. 
     
     
         21 . A real time PCR instrument according to  claim 18 , wherein the reaction vessels comprise capillaries. 
     
     
         22 . A real time PCR instrument according to  claim 21 , wherein the capillaries are glass capillaries having an outer diameter of less than 5 mm. 
     
     
         23 . A real time PCR instrument comprising:
 One optical part for detecting fluorescence emission from a plurality of reaction vessels, the one optical part consisting of:
 an excitation unit comprising:
 at least 1 light source capable of emitting light toward the reaction vessels containing fluorescent compounds, 
 a lightpipe being arranged for receiving light from the reaction vessel and capable of distributing homogeneously said light into optical fiber bundles, wherein the light does not pass through a wavelength excluding device prior to being distributed into said optical fiber bundles, 
 
 a detection unit comprising at least 5 separate fluorescent detector entities, each of said detector entities having a central detection wavelength, said wavelengths being distinct from each other by at least 25 nm, 
 a plurality of at least 5 optical fiber bundles, each of said bundle being arranged for-receiving homogeneously distributed light from the lightpipe, and transmitting said light to said fluorescent detector entities, and 
   means for heating and cooling,   wherein the excitation and detection units are located in separate housings inside the one optical part enabling highly precise positioning of the excitation unit towards each of the reaction vessels without moving the detection unit.   
     
     
         24 . A real time PCR instrument according to  claim 23 , comprising one light source. 
     
     
         25 . An instrument according to  claim 23 , 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. 
     
     
         26 . A real time PCR instrument according to  claim 23 , wherein the reaction vessels comprise capillaries. 
     
     
         27 . A real time PCR instrument according to  claim 26 , wherein the capillaries are glass capillaries having an outer diameter of less than 5 mm.