System for rapid nucleic acid amplification and detection

Abstract

A system is provided for carrying out rapid nucleic acid amplification or other biological reactions requiring thermal cycling. The system of this invention incorporates at least two heating blocks, each having a groove for receiving a reaction vessel such that only a portion of the outer surface of the walls of the vessel are in direct contact with the heating block. The remaining portion of the outer surface of the walls of the reaction vessel remains exposed to ambient conditions. The reaction vessel comes into contact with only one heating block at a time either by movement of the vessel between the heating blocks or by movement of the heating blocks in relation to the vessel. The system of this invention provides rapid temperature cycling without the need for extended ramping times generally associated with single block designs, which include a single temperature block that is forced to heat and cool. Heating and cooling of the reaction using the system of the present invention is accomplished by the reaction vessel and heating blocks coming into thermal contact and reaching thermal equilibrium. The entire vessel need not be surrounded by the heating block, with at least one side of the vessel partially open to ambient conditions. As a result of the configuration of this system it is readily combined with detection systems, for example, fluorescence detection systems.

1 . In a thermal cycling process for polymerase chain reaction (PCR) wherein a reaction vessel (RV) having a vessel wall—containing a reaction mixture comprising a target nucleic acid (NA) and reagents selected to achieve amplification of said target NA by means of the PCR—is thermally cycled between at least two predetermined temperatures, the improvement characterized by maintaining a temperature gradient within said RV during said thermal cycling process. 
     
     
         2 . The process defined in  claim 1 , wherein said temperature gradient is obtained by applying heat unequally to the wall of said RV. 
     
     
         3 . The process defined in  claim 2  wherein said heat is applied unequally to said vessel wall by contacting only a portion of said wall with a heating element while exposing the remainder of said wall to ambient air. 
     
     
         4 . The process as defined in  claim 1 , further characterized by rapid thermal cycling wherein a temperature spectrum of said NA and reagents is maintained within said RV during the thermal cycling process. 
     
     
         5 . The process as defined in  claim 1 , further characterized by annealing and extension of said NA occurring in a single step during the PCR process. 
     
     
         6 . The process as defined in  claim 1 , further characterized by RV temperature ramp-time in excess of 2° C./sec. 
     
     
         7 . The process as defined in  claim 1 , further characterized by real time fluorescence-based measurement of said NA amplification, if present in said reaction mixture, during the PCR process. 
     
     
         8 . The process as defined in  claim 1  wherein said temperature gradient consists of a large gradient. 
     
     
         9 . A polymerase chain reaction (PCR) device for thermal cycling of a reaction mixture contained in a reaction vessel (RV) having an outer wall, comprising at least two heating sources, means for maintaining said heating sources at different predetermined temperatures, and means for causing said RV to be in successive physical contact with each said heating sources for predetermined times and for a number of successive contacts sufficient to promote the PCR in said reaction mixture contained in said RV, characterized by said heating sources being configured for successively contacting only part of said outer wall of the RV while the remainder of said surface is exposed to ambient air. 
     
     
         10 . The PCR device as defined in  claim 8 , wherein said at least two heating sources comprise spaced apart heating blocks each having a groove contoured to receive and provide thermal contact with only a portion of the outer wall of the RV that contains the reaction mixture. 
     
     
         11 . The PCR device as defined in  claim 8 , further comprising means for real-time florescence-based measurement of a preselected nucleic acid (NA), if present in said reaction mixture, during operation of the PCR device. 
     
     
         12 . The PCR device defined in  claim 8  further comprising a controlling means for operating said device so as to achieve a time versus temperature profile which maintains a large temperature gradient within said RV during said thermal cycling process. 
     
     
         12 . The PCR device defined in  claim 11 , wherein said controlling means controls the device to provide annealing and extension of said NA within said RV in a single step during the PCR process by maintaining essentially no holding time at the temperature required for said annealing and extension step. 
     
     
         13 . The PCR device defined in  claim 9 , wherein said at least two heating blocks are in spaced apart opposing relation to each other and said grooves oppose each other. 
     
     
         14 . The PCR device defined in  claim 13 , further comprising a holder for retaining said RV such that when engaged within said groove, and drive means for sequentially moving said RV between said blocks for successive contact with said opposing grooves. 
     
     
         15 . The device defined in  claim 9 , wherein said grooves are dimensioned to contact between about 40% and 50% of the wall of said RV.