Oligonucleotide synthesis with template

For mutagenesis to introduce the stop codons, the protocols in Subheadings Oligonucleotide Phosphorylation with T4 Polynucleotide Kinase, Annealing of the Oligonucleotide to the Template, and step 1 of Enzymatic Synthesis of CCC-dsDNA can be scaled down by a factor of ten. The reaction product can be used directly to transform E. coli using any standard procedure. 

The present study, the template-directed oligonucleotide synthesis, showed a considerable selection of monomers with the aid of such specific base-base interaction. In the preceding trials for template-directed oligonucleotide syntheses, neutral or anionic polynucleotides or polynucleotide analogs were utilized as template molecules However, because of the low affinity between template molecules and mononucleotides, owing to a disadvantage of... 

The most essential point for the template-directed oligonucleotide synthesis is the selective interaction of monomers with the template molecules. As was discussed in 2-1., water-soluble cationic polynucleotide analogs interacted significantly with mononucleotides at relatively low base concentration (-10 > 10 Vol dm ) and the mode of the base-base interaction was dominantly the vertical stacking. In contrast, the base-base interaction in the heterogeneous... 

DNA polymerase enzymes all synthesize DNA by adding deoxynucleotides to the free 3 -OH group of an RNA or DNA primer sequence. The identity of the inserted nucleotide is deterrnined by its abiHty to base-pair with the template nucleic acid. The dependence of synthesis on a primer oligonucleotide means that synthesis of DNA proceeds only in a 5%o V direction if only one primer is available, all newly synthesized DNA sequences begin at the same point. 

The RNA oligonucleotides are complementary to a sequence on one of the strands of the DNA template and base pair with a portion of the DNA molecule. Subsequently, deoxyribonucleotides are covalently attached to the RNA primer. The synthesis of the primer itself is catalyzed by a special RNA polymerase called primase. Similar RNA polymerase-like enzymes are used to prime the synthesis of certain viral DNAs and eukaryotic DNA. 

The RNA world hypothesis requires a nucleotide polymer to reproduce itself. Can a ribozyme bring about its own synthesis in a template-directed manner The self-splicing rRNA intron of Tetrahymena (Fig. 26-26) catalyzes the reversible attack of a guanosine residue on the 5 splice junction (Fig. 26-37). If the 5 splice site and the internal guide sequence are removed from the intron, the rest of the intron can bind RNA strands paired with short oligonucleotides. Part of the remaining intact intron effectively acts as a template for the... 

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