Methods analyzing nucleotide sequences based on their complementarity enable direct analysis of genetic features. Thus, such methods are highly useful for diagnosing genetic diseases, canceration, microorganisms, etc. In addition, methods for amplifying nucleotide sequences, such as PCR, may be applied as such methods enable detection with high sensitivity without any procedures, unlike cell culturing that requires a lot of time and trouble.
Various methods are reported for detecting hybridization of complementary nucleotide sequences. The most fundamental reaction principle widely used is the Southern blot analysis, wherein a sample DNA is immobilized on a nitrocellulose filter to be reacted with a labeled probe. When the sample DNA contains a nucleotide sequence complementary to the probe, the labeled probe is trapped on the nitrocellulose filter through hybridization. A probe for trapping sample DNAs can be used to save the trouble of immobilizing the sample DNAs. In such cases, the labeled probe is trapped in the following order: [solid phase]-[trap probe]-[sample DNA]-[labeled probe]. Regardless of the type of method to be used, a common problem is the adsorption of labeled probes to solid phase independent of the target nucleotide sequence. The non-specific adsorption of labeled probes is the major factor for decreased detection sensitivity. Thus, hybridizations are typically performed in a reaction solution comprising a large quantity of carriers with unrelated nucleotide sequences. Further, the influence of non-specific adsorption is suppressed by thoroughly conducting post-hybridization washing. However, these measures are not sufficient.
Furthermore, methods for analyzing nucleic acids, which do not require the separation of unhybridized labeled probes from the target, are known in the art. For example, a method for detecting homogeneity based upon the difference in the signal of fluorescent label, which changes due to the state of the chains (i.e., single- or double-stranded nucleic acids), is of practical use. The sensitivity of the method is affected by the background signal. Therefore, the problem of the method is that high sensitivity is difficult to achieve with this method. Thus, the method is typically used only when the DNA to be analyzed has been pre-amplified by methods such as PCR.
On the other hand, with the progress of the Human Genome Project, attention has been paid to single nucleotide polymorphism (hereinafter abbreviated as SNP) due to the possibility that the difference of the therapeutic effect of a drug including side effects, and the presence or absence of predisposition to various diseases may be explained by SNP. For example, once a serious side effect of a drug is affected by genetic predisposition based upon SNP, accidents due to the administration of the drug can be avoided by analyzing patients for the presence of SNP associated with the side effect. The term “SNP” refers to a single nucleotide polymorphism in nucleotide sequences of the genome. The human genome, which comprises 3 billion nucleotides, has been suggested to contain approximately one SNP every 1000 nucleotides. The term “SNP” literally refers to a single-nucleotide difference in the genome. As a mater of fact, highly advanced technologies are required to precisely detect single-nucleotide differences. Two nucleic acids having nucleotide sequences complementary to each other hybridize even when the two sequences are not perfectly complementary to each other. Therefore, direct detection of single nucleotide differences by the hybridization method as described above has been found to be difficult.
Currently, the method for detecting known SNPs includes the PCR-SSCP method. This method requires isolation by electrophoresis and thus is not suitable for treating subjects on a large-scale. Hence, a new SNP-detection method without such a drawback is required in the art.
A phenomenon wherein a nucleic acid with specific structure specifically binds to proteins and such based on a different principle from that of hydrogen-bond formation between complementary nucleotide sequences has also been known in the art. Such affinity of the nucleic acid is utilized in the SELEX method (systematic evolution of ligands by exponential enrichment) for selecting nucleic acid molecules with higher affinity in vitro (Nature 355, 564-566, 1990). A nucleic acid with a high binding affinity for a ligand can be obtained by the SELEX method through a repetitive reaction that comprises the steps of contacting the ligand with a library of RNAs having random nucleotide sequences, then recovering the RNAs bound to the ligand, amplifying the recovered RNAs by RT-PCR, transcribing RNAs using the amplified and purified products as templates, and contacting the transcribed RNAs to the ligand. However, it is not generally known that such nucleic acid molecules with selected affinity can be used in various hybridization assays. Particularly, no report exists that indicates the possibility that such nucleic acid molecules are applicable in the detection of SNPs, wherein discrimination of a single nucleotide is required.