Nucleic Acid Chemistry:- The Chemical Synthesis of DNA Has Been Automated
Another technology that has paved the way for many biochemical advances is the chemical synthesis of oligonucleotides with any chosen sequence. The chemical methods for synthesizing nucleic acids were developed primarily by H. Gobind Khorana and his colleagues in the 1970s. Refinement and automation of these methods have made possible the rapid and accurate synthesis of DNA strands. The synthesis is carried out with the growing strand attached to a solid support (Fig. 8–38), using principles similar to those used by Merrifield in peptide synthesis (see Fig. 3–29). The efficiency of each addition step is very high, allowing the routine laboratory synthesis of polymers containing 70 or 80 nucleotides and, in some laboratories, much longer strands. The availability of relatively inexpensive DNA polymers with predesigned sequences is having a powerful impact on all areas of biochemistry (Chapter 9).
FIGURE 8–37 Strategy for automating DNA sequencing reactions. Each dideoxynucleotide used in the Sanger method can be linked to a fluorescent molecule that gives all the fragments terminating in that nucleotide a particular color. All four labeled ddNTPs are added to a single tube. The resulting-colored DNA fragments are then separated by size in a single electrophoretic gel contained in a capillary tube (a refinement of gel electrophoresis that allows for faster separations). All fragments of a given length migrate through the capillary gel in a single peak, and the color associated with each peak is detected using a laser beam. The DNA sequence is read by determining the sequence of colors in the peaks as they pass the detector. This information is fed directly to a computer, which determines the sequence.
FIGURE 8–38 Chemical synthesis of DNA. Automated DNA synthesis is conceptually similar to the synthesis of polypeptides on a solid support. The oligonucleotide is built up on the solid support (silica), one nucleotide at a time, in a repeated series of chemical reactions with suitably protected nucleotide precursors. 1 The first nucleoside (which will be the 3 end) is attached to the silica support at the 3 hydroxyl (through a linking group, R) and is protected at the 5 hydroxyl with an acid-labile dimethoxytrityl group (DMT). The reactive groups on all bases are also chemically protected. 2 The protecting DMT group is removed by washing the column with acid (the DMT group is colored, so this reaction can be followed spectrophotometrically). 3 The next nucleotide is activated with a diisopropylamino group and reacted with the bound nucleotide to form a 5,3 linkage, which in step 4 is oxidized with iodine to produce a phosphotri ester linkage. (One of the phosphate oxygens carries a cyanoethyl protecting group.) Reactions 2 through 4 are repeated until all nucleotides are added. At each step, excess nucleotide is removed before addition of the next nucleotide. In steps 5 and 6 the remaining protecting groups on the bases and the phosphates are removed, and in 7 the oligonucleotide is separated from the solid support and purified. The chemical synthesis of RNA is somewhat more complicated because of the need to protect the 2 hydroxyl of ribose without adversely affecting the reactivity of the 3 hydroxyl.
