Inosine is a free amino group analogues (I), guanosine, at the 2-position of the purine ring. By inosine thing, contributing to the degeneracy of the genetic code, includes a base portion of the coupling of the interaction of codon anticodon A, C, and U,, are found in RNA. It is mutagenic and potentially inosine, rare in DNA caused by deamination of deoxyguanosine.
The number of double-stranded DNA containing inosine, in an attempt to explain the mutagenic effects, and analyzed by X-ray crystallography. Mismatch of · T, I is I · base pairs, shows the pairing error diversity-based G same is assumed the same configuration as the wobble mismatch G · T. That the mutagenicity of inosine can be explained only by reason of the structure, and therefore, it seems possible to be low. Has a thermodynamic stability higher inconsistencies including some inosine surprisingly, when it is possible to repair several enzymes is detected by at least a solid mismatch possibly (and destruction of the base pair and double unstable to be an important factor that recognizes mismatches by insertion of amino acid residues).
Mismatch gives openings or localized melting of the double helix to destabilize the double-stranded DNA generally, to facilitate the flip-base, which when recognizing lesions mutagenic DNA repair enzymes This is one mechanism to be used for. Thus, in this respect, identifying the inosine-containing mismatch is particularly difficult.
I have used the special properties of the inosine-containing mismatches in hybridization probe design based on knowledge of molecular biology, protein sequences. Inosine, incorporated into the nucleotide position of the series of ambiguity due to the degeneracy of the genetic code is present. High thermal stability inosine-containing gap to ensure that the oligonucleotide will hybridize efficiently to the target nucleic acid.
Stability of inosine-containing mismatches, two guanine, guanine-containing base pair mismatches perhaps as compared with guanine counterparts – is explained by the destabilizing effect of the amino group. In the Watson-Crick G · C base pairs stable many, guanine, 2 – amino group, and two water molecules surrounding – forming a hydrogen bond with oxygen in hydrogen bonding and other cytosine. However, (for example in Figure 4, anti-anti-anti-G · blue and G ·), 2 in guanine-containing mismatch specific – because there are more amino group, the G, one is a hydrogen bond completely sterically hindered water fewer hydrogen bonds double-stranded, and discrepancies such is unstable compared to the direction. Destabilization such as do not exist, the 2 – amino group is not present in the inosine.
Easy to use simple, cost-effective, the present invention provides a method and apparatus for the analysis of multiple identification and detection in a sample is fast. In particular, the total time of the sample preparation, the amplification of nucleic acid sequences and differentiation nucleic acid sequence is about 5 hours or less. The method of the present invention, rapid various types of amplifying a nucleic acid sequence using extraction buffer without means for rapid sample processing, nucleic acid compositions unique (II), multi-analyte first amplification step includes (I) of test material means, at the same time, when using appropriate oligonucleotide primers that are optimized to achieve the amplification efficiency similar to non-preferred substantially present in the sample, the target nucleic acid sequence a plurality of Expand, means and mismatch for detecting a nucleic acid sequence that is amplified comprise the recognition nucleic acid (III) process multiple samples, to distinguish, using the appropriate oligonucleotide probe modified with base substitutions neutral molecules Te, the nucleic acid sequence is amplified including a plurality of single base mismatches, detection means for distinguishing the slight differences between the sequences. The product of the first process according to the present invention may comprise a part of the unit or only all.