[Application of polymer detection systems of Histofine MOUSESTAIN, Mouse MAX-PO and rat MAX-PO for mouse and rat frozen tissue sections].

We defined the staining applicability of Histofine MOUSESTAIN, Mouse MAX-PO and Rat MAX-PO for fixed frozen and fresh frozen tissue sections besides paraffin embedded tissue sections. Two different types of cool air dried tissue sections, fresh frozen tissues and fixed frozen tissues of nine-week old Slc: ICR mouse and nine-week old Slc: SD rat, were prepared for immunohistochemistry (IHC) staining in accordance with conventional method. Additional two steps of 1 and 2 to our recommended procedure for paraffin embedded tissue sections were examined as follows: STEP 1: Adjust reaction time of polymer; STEP 2: Add 0.

Arginine-modified DNA aptamers that show enantioselective recognition of the dicarboxylic acid moiety of glutamic acid.

We have screened glutamic acid-binding aptamers from a modified DNA pool containing arginine residues using the method of systematic evolution of ligands by exponential enrichment (SELEX). Thirty-one modified DNA molecules were obtained from the enriched pool after the 17th round of selection, and their binding affinities for the target were evaluated by binding assays using affinity gels. Three modified DNA molecules having higher affinity were sequenced and we determined their affinity and specificity for the target by surface plasmon resonance (SPR) measurements.

Screening of a glutamic acid-binding aptamer from arginine-modified DNA library.

A binder that is specific for a target will be applicable to detection of a variety of molecules such as bioactive substances, environmental contaminants and so on. Nucleic acid aptamers obtained by in vitro selection method are interesting and promising binders. To enhance the ability of nucleic acid aptamers, we prepared a combinatorial modified DNA library containing arginyl uracil base by using arginyl dUTP, then attempted screening of glutamic acid-binding aptamers from the library by in vitro selection method.

Screening of modified DNA aptamers that recognize DNA secondary structure.

We attempted to utilize in vitro selection techniques to develop modified DNA aptamers that are specific for DNA secondary structure including a single-mismatch base pair. For the selection, two kinds of random modified DNA library were prepared by an enzymatic method using modified 2'-deoxynucleoside 5'-triphosphates and KOD Dash DNA polymerase: one library consists of modified DNA including amino functionality and another consists of modified DNA including both hydroxypropynyl and guanidium functionalities.

Systematic characterization of 2'-deoxynucleoside- 5'-triphosphate analogs as substrates for DNA polymerases by polymerase chain reaction and kinetic studies on enzymatic production of modified DNA.

We synthesized C5-modified analogs of 2'-deoxyuridine triphosphate and 2'-deoxycytidine triphosphate and investigated them as substrates for PCRs using Taq, Tth, Vent(exo-), KOD Dash and KOD(exo-) polymerases and pUC 18 plasmid DNA as a template. These assays were performed on two different amplifying regions of pUC18 with different T/C contents that are expected to have relatively high barriers for incorporation of either modified dU or dC. On the basis of 260 different assays (26 modified triphosphates x 5 DNA polymerases x 2 amplifying regions), it appears that generation of the full-length PCR product depends not only on the chemical structures of the substitution and the nature of the polymerase but also on whether the substitution is on dU or dC.

Expansion of repertoire of modified DNAs prepared by PCR using KOD Dash DNA polymerase.

Thymidine analogues bearing a variety of functional groups at the C5-position via an amino-linker arm were prepared and the substrate activity for PCR using thermophilic KOD Dash DNA polymerase was examined. The enzyme accepted the thymidine analogues bearing pyridine, imidazole, biotin, a cationic-charged guanidinium, a cationic-charged amino, mercaptopyridyl and phenanthrolne groups at the C5-position, forming the corresponding PCR product. However, a thymidine analogue bearing a carboxyl group at the C5-position was a poor substrate and the corresponding PCR products could not be obtained.