Methods and Apparatus for Amplifying Nucleic Acids

Abstract

The present invention relates to methods and apparatuses for amplifying, detecting, and optionally quantifying, nucleic acids. In one aspect the method comprises (a) providing a reaction volume comprising (i) a first electrode comprising an electrochemically-active conducting polymer, a first single-stranded nucleic acid molecule capable of hybridizing to a target nucleic acid, wherein the first nucleic acid molecule is covalently attached to the electrochemically-active conducting polymer, and (ii) a second electrode, (b) providing a reaction mixture to the reaction volume, the reaction mixture comprising a target nucleic acid, a nucleic acid polymerase, a redox couple, and nucleic acid amplification reagents, (c) amplifying the nucleic acid, and (d) measuring the impedance of the first electrode at least once during the nucleic acid amplification reaction.

1 .- 14 . (canceled) 
     
     
         15 . An apparatus for real-time nucleic acid amplification, the apparatus comprising
 a) a reaction volume comprising   (i) a first electrode comprising an electrochemically-active conducting polymer, wherein the electrochemically-active conducting polymer is capable of being covalently bound by a single stranded nucleic acid molecule,   (ii) optionally a first single-stranded nucleic acid molecule capable of hydridizing to a first portion of a target nucleic acid sequence, wherein the first nucleic acid molecule is covalently attached to the electrochemically-active conducting polymer, and   (iii) a second electrode; and   b) a reaction mixture comprising ferro-ferricyanide and one or more of
 (i) a second single-stranded nucleic acid molecule comprising nucleic acid sequence complementary to a second portion of the target nucleic acid sequence, 
 (iii) a nucleic acid polymerase, and 
 (iv) a supply of reagents for a nucleic acid amplification reaction 
   
       wherein the reaction volume is suitable for containing a sample comprising nucleic acid, and wherein the reaction volume includes a heater or is adapted to engage with a thermocycler suitable for PCR. 
     
     
         16 . The apparatus of  claim 15  additionally comprising a thermocycler suitable for PCR. 
     
     
         17 . The apparatus of  claim 15  additionally comprising a device for measuring the impedance of at least the first electrode. 
     
     
         18 . The apparatus of  claim 17  wherein the device for device for measuring impedance is an LCR meter or is a potentiostat. 
     
     
         19 . A system for amplifying a target nucleic acid in a sample, the system comprising
 a) a reaction volume comprising
 (i) a first electrode comprising an electrochemically-active conducting polymer, wherein the electrochemically-active conducting polymer is capable of being covalently bound by a single stranded nucleic acid molecule, 
 (ii) optionally a first single-stranded nucleic acid molecule capable of hydridizing to a first portion of a target nucleic acid sequence, wherein the first nucleic acid molecule is covalently attached to the electrochemically-active conducting polymer, and 
 (iii) a second electrode; and 
   b) a reaction mixture comprising ferro-ferricyanide and one or more of
 (i) a second single-stranded nucleic acid molecule comprising nucleic acid sequence complementary to a second portion of the target nucleic acid sequence, 
 (iii) a nucleic acid polymerase, and 
 (iv) a supply of reagents for a nucleic acid amplification reaction. 
   c) a device for measuring the impedance of at least the first electrode; and   d) a thermocycler.   
     
     
         20 . The system of  claim 19 , wherein the device for measuring impedance is an LCR meter, a potentiostat, or the device measures impedance by determining the transconductance of or at the first electrode or by cyclic voltammetry. 
     
     
         21 . The system of  claim 19  wherein the system is used to perform a polymerase chain reaction. 
     
     
         22 . The system of  claim 21  wherein the system is used to measure the impedance of the first electrode at least once during the polymerase chain reaction. 
     
     
         23 . The system of  claim 19  wherein the reaction mixture comprises the first single-stranded nucleic acid molecule, or a single-stranded nucleic acid molecule capable of hydridizing to the first portion of the target nucleic acid sequence. 
     
     
         24 . The system of  claim 22  wherein the system is used to determine the presence or amount of polynucleotide in the reaction volume on the basis of the one or more impedance measurements. 
     
     
         25 . The system of  claim 22  wherein the system is used to measure the impedance of the first electrode before the first elongation step of the polymerase chain reaction. 
     
     
         26 . The system of  claim 22  wherein the impedance is measured continuously throughout at least a portion of the polymerase chain reaction. 
     
     
         27 . The system of  claim 19 , wherein the system is used to measure cumulative charge passed through the electrode. 
     
     
         28 . The system of  claim 22  wherein the system is used to measure cumulative charge passed through the electrode and terminating the polymerisation on the basis of the measurement. 
     
     
         29 . The system of  claim 22  wherein the system is used to measure cumulative charge passed through the electrode and terminating the polymerisation when a total charge of from about 1.0×10 −5  C to about 5×10 −5  C is measured. 
     
     
         30 . The system of  claim 19  wherein the electrochemically-active conducting polymer is selected from the group consisting of electrochemically-active conducting polymers that are or comprise a poly-pyrrole or a derivative thereof, a pyrrolylacrylic acid, poly-3-pyrrolylacrylic acid, pyrrolylbutyric acid, poly-4-(3-pyrrolyl)butyric acid, a co-polymer of poly-pyrrole and 3-pyrrolacrylic acid, a co-polymer of poly-pyrrole and 4-(3-pyrrolylbutyric) acid, a poly-thiophene or a derivative thereof, 3,4-ethylenedioxythiophene, poly(3,4-ethylenedioxythiophene), or a poly-aniline or a derivative thereof. 
     
     
         31 . The system of  claim 30  wherein the electrochemically-active conducting polymer comprises both pyrrole and thiophene. 
     
     
         32 . The system of  claim 18  wherein a target nucleic acid is added to the system to an initial concentration of less than 1 pg/mL. 
     
     
         33 . The system of  claim 32  wherein a target nucleic acid is added to the system to an initial concentration of less than 1 fg/mL. 
     
     
         34 . The apparatus of  claim 16  additionally comprising a device for measuring the impedance of at least the first electrode.