Long, Processive Enzymatic Dna Synthesis ..

An enzymatic method for synthesizing long double-stranded DNA labeled with the halogen derivatives of nucleobases (Hal-NBs) with 1-bp accuracy has been put forward and successfully tested on three different DNA fragments containing the 5-bromouracil (5-BrU) residue. The protocol assumes enzymatic cleavage of two Polymerase-Chain-Reaction (PCR) fragments containing two recognition sequences for the same or different class IIS restriction endonucleases, where each PCR fragment has a partially complementary cleavage site. These sites are introduced using synthetic DNA primers or are naturally present in the sequence used. The cleavage sites are not compatible, and therefore not susceptible to ligation until they are partially filled with a Hal-NB or original nucleobase, resulting in complementary cohesive end formation. Ligation of these fragments ultimately leads to the required Hal-NB-labeled DNA duplex. With this approach, a synthetic, extremely long DNA fragment can be obtained by means of a multiple assembly reaction (n × maximum PCR product length: n × app. 50 kb).

Long, Processive Enzymatic DNA Synthesis Using 100% …

Synthesis of long, representative DNA copies of the murine RNA tumor virus genome.

But synthesising long DNA sequences ..

The real answer lies in the resolution limit of the purification method and on the coupling efficiency of the DNA synthesizer. We can synthesize DNA oligos of 220 bases and obtain sufficient quantities by purification to perform successful gene construction. However, it should be remembered that the longer the oligo, the greater the chance of accumulated sequence errors.

Long DNA strands synthesis optimizing - ResearchGate

All of the DNA synthesis reagents have an impact on good quality DNA synthesis, as are all the steps in the synthesis cycle – coupling, capping, oxidation and detritylation. However, for the successful synthesis of long oligos, some factors are absolutely critical, as noted below.

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The long, precisely labeled DNA duplexes obtained behave in very much the same manner as natural DNA and are beyond the range of chemical synthesis. Moreover, the conditions of synthesis closely resemble the natural ones, and all the artifacts accompanying the chemical synthesis of DNA are thus eliminated. The approach proposed seems to be completely general and could be used to label DNA at multiple pre-determined sites and with halogen derivatives of any nucleobase. Access to DNAs labeled with Hal-NBs at specific position is an indispensable condition for the understanding and optimization of DNA photo- and radio-degradation, which are prerequisites for clinical trials of Hal-NBs in anticancer therapy.

In vitro Synthesis of Long DNA Products in Reactions …

Primer extension assays were performed using a small, circular DNA template to permit unlimited, rolling circle DNA strand displacement synthesis (). Long and processive DNA synthesis after complete replacement of all four unmodified dNTPs by the labeled dN5Ps is shown in . The DNA reached an average length of ∼3000 bases after 5 minutes (∼10 bases/second), compared to ∼4500 bases (∼15 bases/second) with unmodified dNTPs. Longer extension reactions resulted in longer DNA products (data not shown; > 10,000 bases after 20 minutes, beyond the limit of the molecular DNA size marker employed). Agarose gel band intensities were similar compared to the control reaction, indicating unaltered DNA polymerization initiation properties. In contrast, no detectable DNA product was formed when using even just one base-linked dye-labeled nucleotide alongside three unmodified dNTPs (Alexa Fluor 488-7-OBEAdCTP instead of dCTP).

Long Oligos Synthesis | Biolegio

The amount of long cDNA synthesized is maximal at 2 h in 50 muM dNTP; neither longer time nor higher dNTP levels (through 1.8 mM) increased this yield.

TriLink | Long RNA Synthesis, Longmer RNA

In the absence of actinomycin D, equine infectious anemia virus does not require high dNTP levels for either optimal incorporation or long cDNA synthesis.