title: PCR amplification of 4'-thioDNA using 2'-deoxy-4'-thionucleoside 5'-triphosphates.
abstract: 2'-Deoxy-4'-thioribonucleic acid (4'-thioDNA) having a sulfur atom instead of an oxygen atom in the furanose ring has a nuclease resistance and hybridization ability higher than that of natural DNA. Despite its great potential for various biological applications, a long 4'-thioDNA having all four kinds of 2'-deoxy-4'-thionucleosides has not been reported. In this study, we describe systematic analysis of the incorporation of 2'-deoxy-4'-thionucleoside 5'-triphosphates (dSNTPs) using various DNA polymerases. We found that family B DNA polymerases, which do not have 3'→5' exonuclease activity, could efficiently incorporate dSNTPs via single nucleotide insertion and primer extension. Moreover, 104-mer PCR product was obtained even under the conditions in the presence of all four kinds of dSNTPs when KOD Dash DNA polymerase was used. The resulting PCR product was converted into a natural dsDNA by using PCR with dNTPs, and sequencing of the natural dsDNA revealed that the PCR cycle successfully proceeded without losing the sequence information of the template. To the best of our knowledge, this is the first example of accurate PCR amplification of highly modified DNA in the presence of only unnatural dNTPs.
authors: Graduate School of Pharmaceutical Sciences, The University of Tokushima , Shomachi 1-78-1, Tokushima 770-8505, Japan.
authors: Takamitsu
authors: T
authors: Kojima
authors: Kazuhiro
authors: K
authors: Furukawa
authors: Hideto
authors: H
authors: Maruyama
authors: Naonori
authors: N
authors: Inoue
authors: Noriko
authors: N
authors: Tarashima
authors: Akira
authors: A
authors: Matsuda
authors: Noriaki
authors: N
authors: Minakawa
mesh_terms: D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017931:DNA Primers; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D003854:Deoxyribonucleotides / Q000737:chemistry*; D016133:Polymerase Chain Reaction; D058615:Synthetic Biology; D013873:Thionucleotides / Q000737:chemistry / Q000378:metabolism*
chemical_list: D017931:DNA Primers; D003854:Deoxyribonucleotides; D013873:Thionucleotides; D004247:DNA; D004259:DNA-Directed DNA Polymerase