Deoxyribonucleic acid measuring apparatus and method of measuring deoxyribonucleic acid

Abstract

With an insulated gate field effect transistor in which deoxyribonucleic acid (DNA) probes are immobilized on a gold electrode, extension reaction on the gold electrode is performed with DNA polymerase to directly measure an increased amount of a phosphate group caused by the extension reaction, that is, negative charge, by means of a current change between a source and a drain of the insulated gate field effect transistor. Thus, presence/absence of hybridization of target DNAs with the DNA probes, and presence/absence of the extension reaction are detected. Optimum immobilization density of the DNA probes on the gold electrode is set at 4×10 12 molecules/cm 2 . To reduce surface potential fluctuation caused by external variation (influences of foreign substances), which is a problem when using the gold electrode in a solution, a high-frequency voltage equal to or above 1 kHz is applied between the gold electrode and a reference electrode by a power source.

1 . A deoxyribonucleic acid (DNA) measuring apparatus comprising:
 a field effect transistor including a gold electrode contacting with a measurement solution containing DNA polymerase, DNA probes being immobilized on a surface of the gold electrode;   a reference electrode contacting with the measurement solution;   means which applies a high-frequency voltage between the gold electrode and the reference electrode;   means which injects sample DNAs into the measurement solution; and   means which injects at least one type of deoxyribonucleoside triphosphate selected from a group consisting of dATP (deoxyadenosine triphosphate), dCTP (deoxycytidine triphosphate), dGTP (deoxyguanosine triphosphate) and dTTP (deoxythymidine triphosphate), or a deoxyribonucleoside triphosphate derivative.   
     
     
         2 . The DNA measuring apparatus according to  claim 1 , wherein the gold electrode is connected to a gate of the field effect transistor with a conductive wire. 
     
     
         3 . The DNA measuring apparatus according to  claim 1 , wherein a source portion, a drain portion, and a channel portion of the field effect transistor are covered with a light-shielding member. 
     
     
         4 . The DNA measuring apparatus according to  claim 3 , wherein the light-shielding member is a conductive member. 
     
     
         5 . The DNA measuring apparatus according to  claim 3 , wherein the light-shielding member is grounded. 
     
     
         6 . The DNA measuring apparatus according to  claim 3 , wherein the light-shielding member is made of any of aluminum and gold. 
     
     
         7 . The DNA measuring apparatus according to  claim 1 , wherein the high-frequency voltage has a frequency not lower than 1 kHz. 
     
     
         8 . The DNA measuring apparatus according to  claim 1 , wherein at least two sets each consisting of the gold electrode and any of the reference electrode and a pseudo reference electrode are formed on one plane to face each other. 
     
     
         9 . The DNA measuring apparatus according to  claim 1 , wherein the DNA probes are immobilized on the surface of the gold electrode with alkanethiol binding to one end of each of the DNA probes. 
     
     
         10 . The DNA measuring apparatus according to  claim 1 , wherein the deoxyribonucleoside triphosphate derivative is a derivative including an additional portion carrying negative charge which is bound to a side chain of a constituent base. 
     
     
         11 . The DNA measuring apparatus according to  claim 10 , wherein a part of the additional portion contains a phosphate group. 
     
     
         12 . The DNA measuring apparatus according to  claim 1 , wherein the measurement solution contains a deoxyribonucleoside triphosphate decomposing enzyme. 
     
     
         13 . The DNA measuring apparatus according to  claim 1 , wherein the DNA probes contains a base sequence of a universal primer used for sequencing.