1. Field of the Invention
The present invention relates to a method of nucleic acid sequence detection and a nucleic acid sequence detection substrate. Particularly, the present invention relates to a technique for highly sensitively detecting, conveniently and at low cost, the presence of a particular target nucleic acid sequence to be detected in a sample.
2. Description of the Related Art
A conventional method for detecting a nucleic acid sequence specific to a sample as a target involves: binding the target nucleic acid sequence to oligonucleotide strands immobilized on a substrate and binding a different oligonucleotide strand labeled with a fluorescent substance to the bound target nucleic acid sequence; and detecting the presence of the particular nucleic acid sequence in the sample by detecting/amplifying a reflected light signal from the fluorescent substance label by irradiation with light (e.g., laser beam) at a particular wavelength. Another detection method involves: binding the target nucleic acid sequence to oligonucleotide strands immobilized on a substrate; performing elongation reaction using the oligonucleotide strands as primers and the bound target nucleic acid sequence as a template, wherein base substances incorporated during the reaction are labeled with a fluorescent substance; and detecting/amplifying a reflected light signal from the fluorescent substance label by irradiation with light (e.g., laser beam) at a particular wavelength.
However, these detection methods must detect a fluorescent substance and therefore require a special detection apparatus that emits light at a particular wavelength. Such a detection apparatus is expensive and has a limited use to some research institutes or universities under the present circumstances. Thus, these approaches cannot be adopted, for example, for conveniently detecting pathogenic microorganisms such as norovirus or Cryptosporidium in water (sample) at water treatment sites, due to complicated procedures and expensive analysis apparatuses.
Against this backdrop, for example, the MPEX (Multiple Primer EXtension) method described in K. Kinoshita et al., Multiple primer extension by DNA polymerase on a novel plastic DNA array coated with a biocompatible polymer, Nucleic Acid Research, Vol. 35, No. 1, 2007, pp. e3, and Japanese Patent Application Laid-Open Nos. 2006-174788 and 2007-222010 has been developed as a method for conveniently detecting a particular nucleic acid sequence at detection (e.g., water treatment sites) at low cost.
This MPEX method is a genetic testing method using DNA elongation reaction catalyzed by an enzyme (DNA polymerase) on a substrate and is also used in gene mutation analysis, SNP (Single Nucleotide Polymorphism) analysis, and microorganism identification. This method uses, as primers, oligonucleotide strands immobilized on a substrate having, on the surface thereof, a polymer substance containing a first unit having a group induced from phosphoric ester constituting the hydrophilic moiety of phospholipid and a second unit having an active ester group. It involves: after addition of a target nucleic acid sequence, heat-denaturing the double-stranded DNA into single strands on the substrate and performing DNA elongation reaction catalyzed by DNA polymerase using the single strand as a template to incorporate bases, all or some of which are modified with a ligand, into the elongation reaction product (amplified DNA); and finally adding thereto an active substance reactive to the ligand for reaction. The MPEX method thus achieves highly sensitive gene detection.