Method of detecting primer extension reaction, method of discriminating base type, device for discriminating base type, device for detecting pyrophosphate, method of detecting nucleic acid and tip for introducing sample solution

A method of discriminating a base type in which the base type in a base sequence of a nucleic acid is discriminated, said method comprises the following steps of:

• the step (a) of preparing a sample solution containing a nucleic acid, a primer having a base sequence that includes a complementary binding region which complementarily binds to said nucleic acid, and a nucleotide;
• the step (b) of allowing said sample solution to stand under a condition to cause an extension reaction of said primer, and producing pyrophosphate when said extension reaction is caused;
• the step (c) of bringing said sample solution into contact with the front face of a H+ hardly permeable membrane having H+-pyrophosphatase, which penetrates from front to back of the membrane, of which active site that hydrolyzes pyrophosphate being exposed to the front face;
• the step (d) of measuring the H+ concentration of at least either one of the solution at the front face side of said H+ hardly permeable membrane or the solution at the back face side of said H+ hardly permeable membrane, in a state where said H+-pyrophosphatase is immersed in the solution;
• the step (e) of detecting said extension reaction on the basis of the result of measurement in the step (d);
• and
• the step (f) of discriminating the base type in the base sequence of said nucleic acid on the basis of the result of detection in the step (e).

A device for discriminating a base type in which the base type in a base sequence of a nucleic acid is discriminated which comprises:

• a reaction section in which thermoregulation required for an extension reaction of a primer is carried out, and
• a pyrophosphate detection section in which pyrophosphate that is produced by said primer extension reaction is detected, wherein said reaction section is provided with a reserving region for reaction where a solution is reserved,
• said pyrophosphate detection section is provided with a reserving region for detection where a solution is reserved, a H+ hardly permeable membrane that separates said reserving region for detection to a first region and a second region, and a measurement means for measuring the H+ concentration of the solution reserved in at least either one of the first region and second region, and
• wherein said H+ hardly permeable membrane has H+-pyrophosphatase, which penetrates from front to back of the membrane, of which active site that hydrolyzes pyrophosphate being exposed to the front face, and
• in said pyrophosphate detection section, the reaction solution which is delivered from said reaction section is reserved in the first region.

A method of detecting a nucleic acid having a particular base sequence, said method comprises the following steps of:

• the step (a) of preparing a sample solution containing a sample, a primer having a base sequence that includes a complementary binding region which complementarily binds to said nucleic acid, and a nucleotide;
• the step (b) of allowing said sample solution to stand under a condition to cause an extension reaction of said primer, and producing pyrophosphate when said extension reaction is caused;
• the step (c) of bringing said sample solution into contact with the front face of a H+ hardly permeable membrane having H+-pyrophosphatase, which penetrates from front to back of the membrane, of which active site that hydrolyzes pyrophosphate being exposed to the front face;
• the step (d) of measuring the H+ concentration of at least either one of the solution at the front face side of said H+ hardly permeable membrane or the solution at the back face side of said H+ hardly permeable membrane, in a state where said H+-pyrophosphatase is immersed in the solution;
• the step (e) of detecting said extension reaction on the basis of the result of measurement in the step (d);
• and
• the step (f) of detecting the nucleic acid on the basis of the result of detection in the step (e).