Oligonucleotide synthetic

Scheme 1. Automated Synthesis of Oligonucleotides. Synthetic Cycle for the Phosphoroamidite Method.

The deprotected oligonucleotide synthetic product is precipitated twice in ethanol, and a 0.5 fig/fd solution in water is prepared (concentration is measured from a UV absorption spectrum). One microliter of the oligo-deoxynucleotide solution is mixed with 2 fd of 10X PL, 5 fd of [y-32P]ATP (or [y-35S]ATP), 1 fd of T4 polynucleotide kinase, and 11 fd water. After incubation at 37 ° (for 45 min with [y-32P]ATP or for 2 hr with [y-35S]ATP), the reaction is stopped by the addition of 150 [A of 5 M ammonium acetate, pH 5.5, and 130 fd water and 10 fd of the yeast tRNA solution are added to the mixture before precipitation with 1 ml ethanol. After chilling at —70° for at least 15 min, the precipitate is collected by centrifugation (12,000 g, 15 min), redissolved, and submitted to two additional cycles of precipitation-redissolution. Finally, the precipitate is redissolved in 20 fd of gel loading mix and the mixture analyzed on a 8% acrylamide-7 Af urea slab gel in IX electrophoresis buffer, until the bromphenol blue has reached the middle of the gel. 

This was introduced in 1988 principally by Karas and Hillenkamp [19-21], It has since become a widespread and powerful source for the production of intact gas-phase ions from a broad range of large, non-volatile and thermally labile compounds such as proteins, oligonucleotides, synthetic polymers and large inorganic compounds. The use of a MALDI matrix, which provides for both desorption and ionization, is the crucial factor for the success of this ionization method. The method is characterized by easy sample preparation and has a large tolerance to contaminantion by salts, buffers, detergents, and so on [22,23],... 

CPG = Controlled Pore Glass (Solid Support) 1 and 2 (B], 62, 63, 64) Commercially available Scheme 1. Automated Synthesis of Oligonucleotides. Synthetic Cycle for the Phosphoroa-midite Method. 

Although polystyrene-type resins possess chemical stability and ease of functionalization, there is agreement among oligonucleotide synthetic chemists that styrene-based polymers are not compatible with the synthesis of oligonucleotide chains larger than a tri- or tetranucleotide. 

Key words In vivo DNA assembly, Yeast transformation. Gene synthesis. Oligonucleotides, Synthetic... 

Synthetic oligonucleotides may be used as "primers and be elongated stepwise with the aid of polynucleotide phosphorylase (PNPase) and nucleoside diphosphates. 

Each primer is a synthetic oligonucleotide of about 20 bases prepared so that then-sequences are complementary to the (previously determined) sequences that flank the tar get regions on opposite strands Thus one primer is annealed to one strand the other to the other strand The 3 hydroxyl end of each primer points toward the target region The stage is now set for DNA synthesis to proceed from the 3 end of each primer [Figure 28 14(c )] The solution contains a DNA polymerase and Mg " m addition to the... 

Fig. 9. Mutagenesis by a synthetic oligonucleotide of a cloned sequence available in single-stranded form (a) single-stranded M13 template containing uracil (U) residues (b) double-stranded product, uracil residues are not mutagenic (c) strong selection for M13 phages containing mutation of interest (23).

Phosphorothioates. All three synthetic approaches appHcable to unmodified oligonucleotides can be adapted for synthesis of phosphorothioates (11) (33,46). If all of the phosphodiester linkages in an oligonucleotide are to be replaced with phosphorothioates, the ff-phosphonate method for coupling, followed by oxidation with Sg in carbon disulfide and triethylamine in the final step, is the most straightforward method. 

Moreover, disposable electrochemical sensors for the detection of a specific sequence of DNA were realised by immobilising synthetic single-stranded oligonucleotides onto a graphite or a gold screen-printed electrode. Tire probes became hybridised with different concentrations of complementary sequences present in the sample. 

A number of standard synthetic reference books are available. A review article by Kossell and Seliger discusses protective groups used in oligonucleotide syntheses, including protection for the phosphate group, which is not included in this book, and a series of articles describe various aspects of protective group chemistry. 

Synthetic organic polymers, which are used as polymeric supports for chromatography, as catalysts, as solid-phase supports for peptide and oligonucleotide synthesis, and for diagnosis, are based mainly on polystyrene, polystyrene-divinylbenzene, polyacrylamide, polymethacrylates, and polyvinyl alcohols. A conventional suspension of polymerization is usually used to produce these organic polymeric supports, especially in large-scale industrial production. 

Phosphate esters and anhydrides dominate the living world. Major areas of synthetic interest include oligonucleotides (polymeric phosphate diesters), phospho-rylated peptides, phospholipids, glycosyl phosphates, and inositol phosphates. ... 

Preorganization of DNA and improving nucleic acid recognition by synthetic oligonucleotides 97CRV1473. 

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