By Douglas J. Dellinger, Jason R. Betley, Tadeusz K. Wyrzykiewicz (auth.), Piet Herdewijn (eds.)
Nucleic acids chemistry has advanced swiftly within the final 10 years with the creation of many new options and the advance of many novel healing entities. In Oligonucleotide Synthesis: equipment and purposes, laboratory specialists describe in step by step element the strong new recommendations they've got constructed for oligonucleotide synthesis and for using changed oligonucleotides in biotechnology. one of the protocol highlights are the synthesis of phosphorothioates through new sulfur move brokers, the synthesis of LNA, peptide conjugation tips on how to enhance mobile supply and cell-specific focusing on, triple helix formation, and a singular two-step approach that yields a excessive purity DNA that's more cost-effective. Novel functions contain using molecular beacons to watch the PCR amplification technique, nuclease footprinting to review the sequence-selective binding of small molecules to DNA, nucleic acid libraries, optimization of RNA synthesis for siRNA purposes, and using small interference RNA (siRNA) as an inhibitor of gene expression. The protocols keep on with the profitable tools in Molecular Biology™ sequence layout, each providing step by step laboratory directions, an creation outlining the primary in the back of the strategy, lists of the required apparatus and reagents, and tips about troubleshooting and warding off identified pitfalls.
state-of-the-art and hugely functional, Oligonucleotide Synthesis: tools and functions bargains brand new investigators not just perception into vital new biotechnologies, but additionally a whole variety of the targeted protocols had to paintings effectively in DNA synthesis and its purposes today.
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Additional resources for Oligonucleotide Synthesis
Store solution at –20°C. Fresh reagent should be prepared weekly. 8. 18 mL perchloric acid in HPLC-grade water, and bring the volume up to 1 L using HPLC-grade water. 9. HPLC buffer B:. 18 mL perchloric acid in HPLC-grade water, and bring the volume up to 1 L using HPLC-grade water. 10. 44 g EDTA in molecular-grade water to a final volume of 4 L. 11. 1X TBE electrophoresis buffer: Bring 200 mL of 5X TBE up to 1 L with molecular-grade water. 12. 8% polyacrylamide 7M urea stock solution: Mix 400 mL of 40% acrylamide solution (29:1 acrylamide:bis) with 400 mL 5X TBE buffer, dissolve 841 g urea into the solution, and dilute to 2 L with molecular-grade water.
23. Hi-Prep 26/10 desalting column (Amersham Biosciences). 24. 6 × 250 mm (Waters). 25. HPLC-grade acetonitrile. Toxic. 26. Ammonium bicarbonate. 27. Glacial acetic acid. Caution: Corrosive. 3. Methods The methods outlined below describe: (1) the solid-phase synthesis of oligoribonucleotides, manually or machine aided, (2) the deprotection of the oligoribonucleotides, (3) the purification of oligoribonucleotides, and (4) the analysis of the purified product. 1. 1. 2. For those people without access to a solid-phase synthesizer, the RNA can be synthesized manually with a minimum of equipment.
9. Dichloroacetic acid (DCA) (Aldrich-D5, 470-2). 10. 1M 2'-ACE-Me-phosphoramidite (Dharmacon). 11. 1M Disodium 2-carbamoyl-2-cyanoethylene-1,1-dithiolate in N,N'-dimethyl formamide 1 (Dharmacon) (8). 12. 40% 1-methylamine in water (Aldrich-42, 646-6). 13. 3'-O-linked ribonucleoside on polystyrene support (Dharmacon). 14. 8 (pH with TEMED). 15. N,N,N',N'-tetramethylethylenediamine (TEMED). 16. 0. 17. 0. 18. 0, 10 mM ethylene diamine tetraacetic acid (EDTA). 19. 8 and 15% polyacrylamide 7M urea stock solutions.