ABSTRACT
In order to enhance a collection of modified deoxynucleoside triphosphates useful for in vitro selection or SELEX (systematic evolution of ligands by exponential enrichment) techniques, we designed and synthesized modified analogues of 2'-deoxyuridine triphosphate and 2'-deoxycytidine triphosphate bearing a flexible and hydrophilic 7-amino-2,5-dioxaheptyl linker at a C5 position. Both analogues were found to be substrates for thermostable DNA polymerases which belong to an evolutional family B during PCR.
Subject(s)
DNA-Directed DNA Polymerase/metabolism , Pyrimidine Nucleosides/chemical synthesis , Pyrimidine Nucleosides/metabolism , Base Sequence , DNA/chemistry , Ethidium , Indicators and Reagents , Molecular Sequence Data , Reverse Transcriptase Polymerase Chain Reaction , Sequence Analysis, DNA , Substrate SpecificityABSTRACT
Modified analogs of 2'-deoxycytidine triphosphates bearing (6-aminohexyl)carbamoylmethyl or 7-amino-2,5-dioxaheptyl linker at a C5 position were designed and synthesized. Both analogs were found to be good substrates for Vent(exo-) DNA polymerase during PCR, resulting in the corresponding full-length modified DNAs, respectively. Moreover, we have demonstrated simultaneous incorporation of three different modified nucleotides into a DNA strand by PCR using triphosphates of 5-(3-aminopropynyl)dUTP, 5-[(6-aminohexyl)carbamoylmethyl]dCTP and 2-amino-dATP (dDTP) or N6-methyl-dATP in place of the natural nucleoside triphosphates TTP, dCTP and dATP.
Subject(s)
Nucleotides/chemistry , Polymerase Chain Reaction/methodsABSTRACT
Triphosphate of a new fluorescent labeled thymidine analogue was incorporated as a substrate for PCR using KOD Dash DNA polymerase forming the corresponding fluorescent labeled DNA which is useful for a DNA probe.
Subject(s)
DNA-Directed DNA Polymerase/metabolism , DNA/chemical synthesis , Fluorescent Dyes/metabolism , Nucleotides/metabolism , Thymidine/metabolism , Base Sequence , DNA Probes , Polymerase Chain ReactionABSTRACT
Modified 2'-deoxyuridine triphosphates bearing proteinous amino acids at a C5 position were prepared, and their substrate properties were investigated for KOD Dash DNA polymerase during PCR. The modified dUTPs bearing histidyl, lysyl, glutaminyl or seryl group produced the aimed 108 nt PCR products in good yields. In contrast, the analog bearing glutamyl group did not work as a substrate for KOD Dash while the analog bearing aspartyl group gave the product in a low yield. Moreover, not only KOD Dash but also three other thermostable DNA polymerases were tested as catalysts by use of C5 modified dUTPs with two different types of linker arms. Both Pfu and Vent(exo-) were relatively tolerant for the modification at the C5 position as well as KOD Dash.
