The Chemical -.-yiE2:..::.:E.::.:: ..:__. .a_...... Synthesis of DNA and Its Uses in L to R: Bruce Kaplan, GarrettLarson, and John Rossi Molecular Garrett Larson is a graduate student with a joint appointment in the Molecular Genetics Dept. at the Beckman Research Institute of the City of Hope and the Department of Microbiology at, the Univ of California Los Angeles. He received his B.S. in bio- Biology chemistry from the Univ. of California, Riverside. Bruce Kaplan is an associate research scientist in the Dept. of Mo- lecular Genetics at the Beckman Research Institute of the City of Hope. He received his B.S. from City College of New York and Garrett Larson Downloaded from http://online.ucpress.edu/abt/article-pdf/46/8/440/86223/4447895.pdf by guest on 01 October 2021 his Ph.D. in organic chemistry from Cornell University. Before Bruce E.Kaplan coming to the City of Hope, Dr. Kaplan served as an assistant professor at Cornell Univ. from 1963-1967, at the California State John J. Rossi Univ. at Los Angeles from 1967-1971 and as a research scientist at Rockwell International from 1971-1973. At the City of Hope he was director of the Laboratory of Organic Synthesis from Progress in science is a history of the synergistic 1972-1979. In 1980, he joined the Molecular Genetics Depart- ment to work on the synthesis of oligodeoxyribonucleotides. relationship between technology and theory: John Rossi is presently an assistant research scientist in the Dept. growth in theoretical understanding requires and of Molecular Genetics at the Beckman Research Institute of the initiates technological change; technological ad- City of Hope. He received his B.A. at the Univ. of New vance opens up new areas of knowledge. The histo- Hampshire, and his Ph.D. in 1976 in genetics from the Univ. of Dr. Rossi served as a fellow ry of molecular genetics is but a illustration of Connecticut. postdoctoral research in single the laboratory of Dr. Arthur Landy at Brown Univ. before com- this process. From Mendel's achievement in predict- ing to the City of Hope in 1980. ing the genetic characteristics of future generations to our present day ability to detect within a gene one faulty base in the four billion bases in an entire enzymes was greatly enhanced by the development human genome-scientific understanding of the of sequencing techniques by Fred Sanger and col- gene has had a dramatic and accelerating history, leagues at Cambridge University and later by Alan punctuated by a series of key events. Four of the Maxam and Walter Gilbert at Harvard University. major technological achievements in molecular biol- These techniques enable molecular geneticists to ogy have been the use of X-ray diffraction to posit characterize fragments of DNA and finally to se- a model for DNA, the discovery of DNA cutting quence entire genes. enzymes, the sequencing of DNA, and the chemical Perhaps the most significant development, in its synthesis of DNA. potential for understanding the functions of DNA, Scientists could not begin to answer the question is the recent availability of short, chemically synthe- of how DNA functions without having a three-di- sized, single-stranded DNAs, or oligonucleotides mensional conceptualization of the DNA molecule. (Gr. ligos:few). The use of these oligonucleotides as In 1953, using the results of the technique of X-ray a standard technique in molecular is in its (oignuleotides grasl highlyspcificy geneticsproesfeeorpgene diffraction, Watson and Crick gave "form" to the infancy. All major molecular genetics laboratories DNA molecule, providing a three-dimensional are learning how to these DNA idntfiationd WandeGislaetin prepare andthHability fragments.tonalersiy model of DNA as a self-complementary double he- Chemically synthesized DNA oligonucleotides are lix. The complementary nature of this model indi- now readily synthesized and purified in just two cated an essential aspect of DNA function; that is, it days using automated synthesizers. Their availability serves as a template in ordering amino acids during has made possible many new projects and propa- the biosynthesis of proteins. gated a large number of ingenious experiments Further advances in DNA research have brought yielding valuable scientific information. These in- about the ability to cleave the DNA molecule at clude the synthesis of medically important proteins specific sites using enzymes called restriction endo- using artificially constructed genes (oligonucleotide nucleases and to sequence the resulting fragments. gene assembly), the ability to isolate a specific gene Scientists have isolated many restriction endo- from a cloned library of random DNA fragments nucleases; more than 200 are now commercially available for scientific use. The usefulness of these 440 THE AMERICAN BIOLOGY TEACHER, VOLUME 46, NO. 8, NOVEMBER/DECEMBER 1984 precisely a gene sequence (synthetic DNA mediated reactions can be driven to completion by using a site-specificin vitromutagenesis). large excess of reactants. Two methods are currently being used for the Chemical Synthesis of DNA synthesis of DNA oligonucleotides: the phos- Oligonucleotides B1 The current availability of chemically synthesized DNA oligonucleotides is the result of almost three O-L|OH decades of development of increasingly faster and easier synthetic techniques. From the mid-1950s, when the synthesis of DNA was begun, through the mid-1970s, when many research groups were in- volved in the synthesis of DNA, the techniques for DNA synthesis were too complicated to be accom- plished by anyone but an experienced organic 0 COUPLING chemist. Today, the chemical synthesis of DNA % B2 oligonucleotides has been simplified such that any Downloaded from http://online.ucpress.edu/abt/article-pdf/46/8/440/86223/4447895.pdf by guest on 01 October 2021 biologist, with relatively minimal background in organic chemistry can synthesize them. -o-jOH'LBa + ( P | The solution technique for DNA synthesis, devel- oped by the organic chemist H. Gobind Khorana, which was used in the 1950s through the 1960s, has become known as the phosphodiester method. The -O-LOH term phosphodiester refers to the product molecules 0- P |,-OH which were diester phosphate salts. The purification )Cap unreactedB1, washout of these salts was extremely time consuming and, IurreactedB2, remove5' Bl B2 V p'otectinggroup X since the yields were not very high, each product Bi 1 had to be characterized at every step in the syn- ?0P 0-OH thesis. Bl B2 The first improvement in the synthesis of DNA oligonucleotides became known as the phos- photriestermethod. This method made use of much of the chemistry developed by Khorana but, with several changes, produced triester instead of diester -ol-CAP phosphate salts. The yields of DNA oligonucleotides by the phosphotriester method were higher than by CluRereate a nd? ut the phosphodiester method, the products were much more easily purified by standard chromato- graphic techniques, and the time required for @Cleave completed chains oligonucleotide synthesis was dramaticallyreduced. fromInert resin support Thus, where the phosphodiester method required nearly two days of labor per internucleotide bond Bl (n)(naI) formed, the phosphotriester method required only 0HO-|,P'LPL OH-5 two hours per bond. The next advance in the synthesis of DNA Figure 1. General scheme for the chemical synthesis of DNA oligonucleotides required a shift of the chemistry oligonucleotides using a solid support. from solution methods to solid support (solid phase) The 3' nucleotide is first coupled to the solid support (a poly- methods. After many unsuccessful early attempts to styrene copolymeror controlled pore size glass beads). The solid modify the preexisting techniques of organic pep- support containing the first nucleotide (B1) is coupled with the next protected nucleotide (B2) using a coupling catalyst. Any un- tide synthesis, Michael Gait and Keiichi Itakura fi- reacted 3' nucleotide bound to the solid support is capped with nally succeeded in adapting these techniques to acetic anhydride, and unreacted nucleotides in solution are oligonucleotidesynthesis. The solid phase synthesis is washed out of the reaction vessel. This prevents any undesired side reactions. A protecting group (X) is chemically removed advantageous over solution methods because the from the dinucleotide to give a new reactive site at the 5' end growing oligonucleotide is easily purified from solu- of the growing oligonucleotide chain. Operations 1 and 2 are re- ble reaction by-products. In addition, mechanical peated until the desired sequence is synthesized. After the syn- thesis, all protectinggroups are removed and the final product is losses are virtually eliminated through the use of purified by high performance liquid chromatography or gel only one reaction vessel, and most importantly the electrophoresis . CHEMICAL SYNTHESIS OF DNA 441 photriesterand the phosphoramidite methods. Since a) short oigonucleotides b) long oligonucleotides most of our experience in this laboratory has in- volved use of the phosphotriester method, we will DNA ligase DNA polymerose I confine our discussion to a description of this meth- EcoRl od. It should be pointed out that the phosphora- synthetic gene BamHl EcoRI BomH midite procedure is operationally similar. 1 ~~~~EcoRlI l The solid phase phosphotriester method for the czori ng ~~~~~~~~~~~~BomH synthetic gene synthesis of oligonucleotides (Figure1) consists of the following steps: a) Functionalization of the solid Cloning EcoRl EcoRl EcoRl support (a polystyrene resin) so that the first nu- BomHI synthetic gene synthetic gene EoR cleoside can be attached to the support, b) Attach- AMP Tet BomHi Asrp BomHi tPR322 BomHl tronstormotion \ ment of the first nucleoside to the support, c) Re- moval of one protecting group (a protecting group Amp Tet blocks chemical reactions at the site where they are located) from the attached nucleoside to allow for a Figure 2. Assembly of gene fragments from synthetic DNA oligonucleotides. reactive site to be uncovered, d) Coupling of the (a) Template dependent ligation-In this approach short, com- next nucleoside to the reactive site.