PCR amplification of 4'-thioDNA using 2'-deoxy-4'-thionucleoside 5'-triphosphates. 2013

Takamitsu Kojima, and Kazuhiro Furukawa, and Hideto Maruyama, and Naonori Inoue, and Noriko Tarashima, and Akira Matsuda, and Noriaki Minakawa
Graduate School of Pharmaceutical Sciences, The University of Tokushima , Shomachi 1-78-1, Tokushima 770-8505, Japan.

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.

UI MeSH Term Description Entries
D003854 Deoxyribonucleotides A purine or pyrimidine base bonded to a DEOXYRIBOSE containing a bond to a phosphate group. Deoxyribonucleotide
D004247 DNA A deoxyribonucleotide polymer that is the primary genetic material of all cells. Eukaryotic and prokaryotic organisms normally contain DNA in a double-stranded state, yet several important biological processes transiently involve single-stranded regions. DNA, which consists of a polysugar-phosphate backbone possessing projections of purines (adenine and guanine) and pyrimidines (thymine and cytosine), forms a double helix that is held together by hydrogen bonds between these purines and pyrimidines (adenine to thymine and guanine to cytosine). DNA, Double-Stranded,Deoxyribonucleic Acid,ds-DNA,DNA, Double Stranded,Double-Stranded DNA,ds DNA
D004259 DNA-Directed DNA Polymerase DNA-dependent DNA polymerases found in bacteria, animal and plant cells. During the replication process, these enzymes catalyze the addition of deoxyribonucleotide residues to the end of a DNA strand in the presence of DNA as template-primer. They also possess exonuclease activity and therefore function in DNA repair. DNA Polymerase,DNA Polymerases,DNA-Dependent DNA Polymerases,DNA Polymerase N3,DNA Dependent DNA Polymerases,DNA Directed DNA Polymerase,DNA Polymerase, DNA-Directed,DNA Polymerases, DNA-Dependent,Polymerase N3, DNA,Polymerase, DNA,Polymerase, DNA-Directed DNA,Polymerases, DNA,Polymerases, DNA-Dependent DNA
D013873 Thionucleotides Nucleotides in which the base moiety is substituted with one or more sulfur atoms.
D016133 Polymerase Chain Reaction In vitro method for producing large amounts of specific DNA or RNA fragments of defined length and sequence from small amounts of short oligonucleotide flanking sequences (primers). The essential steps include thermal denaturation of the double-stranded target molecules, annealing of the primers to their complementary sequences, and extension of the annealed primers by enzymatic synthesis with DNA polymerase. The reaction is efficient, specific, and extremely sensitive. Uses for the reaction include disease diagnosis, detection of difficult-to-isolate pathogens, mutation analysis, genetic testing, DNA sequencing, and analyzing evolutionary relationships. Anchored PCR,Inverse PCR,Nested PCR,PCR,Anchored Polymerase Chain Reaction,Inverse Polymerase Chain Reaction,Nested Polymerase Chain Reaction,PCR, Anchored,PCR, Inverse,PCR, Nested,Polymerase Chain Reactions,Reaction, Polymerase Chain,Reactions, Polymerase Chain
D058615 Synthetic Biology A field of biological research combining engineering in the formulation, design, and building (synthesis) of novel biological structures, functions, and systems. Biologies, Synthetic,Biology, Synthetic,Synthetic Biologies
D017931 DNA Primers Short sequences (generally about 10 base pairs) of DNA that are complementary to sequences of messenger RNA and allow reverse transcriptases to start copying the adjacent sequences of mRNA. Primers are used extensively in genetic and molecular biology techniques. DNA Primer,Oligodeoxyribonucleotide Primer,Oligodeoxyribonucleotide Primers,Oligonucleotide Primer,Oligonucleotide Primers,Primer, DNA,Primer, Oligodeoxyribonucleotide,Primer, Oligonucleotide,Primers, DNA,Primers, Oligodeoxyribonucleotide,Primers, Oligonucleotide

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