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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 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 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 of 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 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 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 . 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 , or mensional conceptualization of the DNA . (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 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 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 ( 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 . 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 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 support. from solution methods to solid support (solid phase) The 3' 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 in solution are oligonucleotidesynthesis. The solid phase synthesis is washed out of the reaction vessel. This prevents any undesired side reactions. A (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 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. Operations c plementary oligonucleotides (10-15 bases) are annealed and li- Downloaded from http://online.ucpress.edu/abt/article-pdf/46/8/440/86223/4447895.pdf by guest on 01 October 2021 and d are repeated until the desired oligonucleo- gated in the presence of DNA ligase. tide has been assembled. (b) Overlapping polymerization method-Long oligonucleotide The purification of chemically synthesized (30-40bases) containing complementarysequences at their 3' ter- mini are annealed. After the addition of E. coli DNA polymerase oligonucleotides is a multi-step operation. The and DNA ligase, a complete double-stranded DNA molecule is oligonucleotide is first cleaved from the solid sup- synthesized. port and all of the protecting groups are removed. In both approaches, the synthetic gene fragmentis cleaved with The oligonucleotide solution is then placed on a the appropriaterestriction enzyme (in this example Eco RI and BamHI), ligated into a plasmid vector (pBR322in this case) and chromatographycolumn to remove the bulk of the transformedinto an E. coli host. impurities. The final purificationis carriedout using high performance liquid chromatography (HPLC). The DNA that is to be purified by HPLC is of two nucleotides in length, Figure 2b (Rossi, Kierzek, types: the desired full length oligonucleotide and Huang, Walker and Itakura 1982). Our approach shortersequences which result from undesirable side also takes advantage of the complementary nature reactions. The high resolution obtainable by two of DNA. For the same 100 base pair gene as above, successive HPLC purifications yields the pure full- four oligonucleotides, each 29 bases in length, are length oligonucleotide suitable for biological use. chemically synthesized. These oligonucleotides are The solid phase synthesis of DNA oligonucleotides designed such that each pair has eight bases of com- has become so fast and reliable that it has com- plementary sequence at their 3' terminus. The pletely supplanted the synthesis of oligonucleotides oligonucleotides are mixed in vitro such that the by solution techniques. Solid phase synthesis has also complementary ends form hydrogen bonds with made possible the complete automation of DNA one another; this results in a partially double synthesis, and several machines capableof DNA syn- stranded molecule which is now a for the thesis are now commercially available. bacterial enzyme E. coli DNA polymerase. After a short incubation in the presence of this enzyme and all four deoxynucleoside triphosphates, a complete Assembly of Genes from Synthetic DNAs double stranded molecule is produced. Thus, in- One of the first uses for synthetic DNA was pi- stead of chemically synthesizing and joining 200 nu- oneered by Khorana and his colleagues in the cleotides, we only need to chemically synthesize 116 1970s;they chemically synthesized and assembled a nucleotides (a 40% reduction); the remainder of the gene containing the coding information for a trans- nucleotides are introduced in a matter of minutes fer RNA (Khorana 1979). In this extraordinaryac- by the in vitro enzymatic polymerization reaction. complishment, a series of oligonucleotides 10-15 Both of the above described methods have been bases in length were chemically synthesized and used successfully to assemble genes of medical and joined together in the same manner as outlined in biologicalinterest. Figure 2a. Using this approach for a gene segment One of the major goals for the practical applica- 100 bases pairs long, there is a requirement for the tion of recombinant DNA technology has been to chemical synthesis of 200 nucleotides. We have de- express in bacteria human genes coding for medi- veloped an alternative approach that was made cally importantpeptides. The molecular signals used possible by the improved chemistry, enabling the for efficient expression of genes in the bacteriumEs- chemical syntheses of oligomers of 30 and even 40 cherichiacoli are well characterized. These signals,

442 THE AMERICAN BIOLOGY TEACHER, VOLUME 46, NO. 8, NOVEMBER/DECEMBER 1984 however, are very different from those used in SyntheticDNA as a Highly Specific human gene expression. When a gene is chemically Molecular Probe synthesized, all of the molecular signals required for efficient expression of that gene in an organism The first step in most gene cloning experiments is such as Escherichiacoli can be designed and incorpo- to identify and isolate a particular gene or gene rated into the synthetic sequence. In addition, the fragment from an organism's genome. The diffi- design of the gene can be such that segments can culty of identifying one gene out of millions can be be removed and replaced conveniently using re- easily appreciated. Utilizing the complementary strictionendonuclease recognition sequences. As dia- nature of DNA, scientists are now designing syn- grammed in Figure 2, the synthetic gene must be thetic oligonucleotides that will identify these genes joined to a vector, in this case a bacterial plasmid cloned in bacterial and phage vectors. These allow vector, so that it can be propagated and expressed the detection of a specific bacterial colony or phage in a biological system. The designs of synthetic genes plaque, (out of huge population) which contains are such that insertion into the vector is easily per- the cloned gene of interest. Genes coding for both formed because of judicious choices of restriction normal and mutant proteins have been isolated endonuclease cleavage sites which are incorporated using this technique.

within the synthetic sequence. If the amino acid sequence of a particularprotein Downloaded from http://online.ucpress.edu/abt/article-pdf/46/8/440/86223/4447895.pdf by guest on 01 October 2021 Synthetic genes coding for a wide range of medi- or even just a portion of the sequence is known, cally useful peptides have been created, cloned, then the possible codon combinations for the DNA and expressed in bacterialand yeast host-vector sys- that could code for the protein can be deduced. If tems. These include sequences coding for the pep- we focus on a small segment of the protein, one of tides insulin (Hirose, Kraszewski, Itakuraand Riggs about six amino acids, and choose the DNA se- 1979), growth hormone, somatostatin, alpha inter- quence so as to keep the number of different feron, epidermal growth factor, insulin like growth codons minimized, then a mixture of oligonucleo- factor and perhaps a score of others not yet pub- tides which represent all possible codon combina- Zicized.Already the first synthetic gene to be used tions can be synthesized (Figure 3). This population for commercial purposes, that coding for human in- of oligonucleotides differing at only a few bases is sulin, is making available large quantities of the called a mixed probe. This probe can be used to human version of the widely required hormone. identify the gene that encodes the protein we have For many of these proteins and peptides, the amino isolated. The gene is identified from a bacterial or acid sequence was the only information available; phage library of genomic DNA or a cDNA (DNA that is, no DNA or RNA sequences were deter- copies of mRNAs made using ) mined. By reverse translating the amino acid se- plasmid library(see Jon Geiger's introductoryarticle quence into a genetic code, then chemically syn- in the October issue for a description of libraries). thesizing and assembling this code, the cloning and After the bacterial colonies or phage plaques con- expression of these genes was made possible well taining the library of genes are spread on petri ahead of the time it would have taken to identify, plates containing nutrient agar, the colonies (or sequence, and genetically engineer the natural plaques) are blotted onto filter paper such that via- DNA sequence. ble colonies will remain on the petri plate to pro-

Gene Sequence 5' TGGATG GAG CAG GAG GGG 3' Trp Met Glu Gln Glu Gly AminoAcid Sequence (Double Stranded DNA) 3' ACCTAC CTC GTC CTC CCC 5' 5' UGGAUG GAG CAG GAG GG 3' 8 Possible mRNASequences or or or I transcription A A A + into mRNA 4 A A A mRNASequence 5' UGGAUG GAG CAG GAG GGG 3' or or or 5' TGGATG GAG CAG GAG GG 3' 8 Possible Gene Sequences (Double inpransatein pronlteion | 3' AGGTAG GTG orGAG orGTG orGG 5' Stranded DNA) AminoAcid Sequence Trp Met Glu Gln Glu Gly T T T 4 3' ACCTAC CTC CAC CTC CC 5' Synthetic DNA,a Mixture or or or of 8 Different Sequences T T T

Sequence of Nucleotides in DNASpecifies AminoAcid Sequence in Protein Specifies AminoAcid Sequence in Protein. a Degenerate Set of Nucleotide Sequences.

Figure 3. Strategy for making a mixed synthetic DNA probe based on amino acid sequence data.

CHEMICAL SYNTHESIS OF DNA 443 vide a "master" plate. The filter paper replicas pA pth bearing the bacterial colonies are treated with a. probe b. probe strong alkali, this lyses the bacteria and simul- taneously denatures their DNA. The filters are then placed in a sealed bag together with the ra- dioactively labeled mixed probe in a buffered salt solution. The filters are incubated at a predeter- mined temperature for a number of hours until the radioactively labeled oligonucleotide complemen- tary to the gene of interest has hybridized (base paired) to the cloned gene. After this incubation period, the filters are rinsed in a salt solution to re- move any oligonucleotides binding to noncomple- mentary DNA. After rinsing, the radioactive filters are placed upon X-ray film to obtain an auto- radiograph which identifies clones containing the Downloaded from http://online.ucpress.edu/abt/article-pdf/46/8/440/86223/4447895.pdf by guest on 01 October 2021 gene of interest (positives). The exact clone bearing the gene of interest can be isolated by orientating the autoradiograph to the master plate. 1.8 kb- Mixed probes have been used for the isolation of bacterial clones encoding a number of different mammalian proteins; these include the human his- AA plhplh AA pth13th tocompatibility complex (transplantation antigens) Aplth Aplth and important blood clotting factors. Figure 4. Use of synthetic DNA probe to detect a genetic disease. Further exploiting the complementary nature of Two synthetic 19 base oligonucleotides complementary to the DNA, scientists have begun to use synthetic normal 13-globin (P3A) and thalassemic (13th) 13-globin genes were oligonucleotide probes in the diagnosis of molecular used to screen individuals for the disease. The DNA sequences of normal versus 13-thalassemia specific probes differ by only a single disorders that are caused by a single nucleotide nucleotide as do the gene sequences. change in a gene coding for an important protein. Chromosomal DNAs from normal (AA), thalassemia hetero- Such probes have been used to locate errors in the zygous (Ar3th), and thalassemia homozygous (rlthflth) individuals were cleaved with a restriction endonuclease, fractionated by human genome which cause 3-thalassemmia (Pi- agarose gel electrophoresis, and transferred to nitrocellulose pa- al. and sickle cell anemia. These dis- The is located on a 1800 base (1.8kb) rastu, et 1983) per.fet a f3-globinb rdctdnshr,te.s_fsytei gene pair eases are caused by one nucleotide change in the chromosomalDNs il DNApnordso:ciicldig fragment.pne Radioactively labeled oligonucleo- tide probes either complementary to the normal 13-globin gene DNA coding for the 3-globin gene. To identify this noses.:_ sequence (P3A), or complementary to the li-thalassemia gene se- one base error in the human genome is to look for quence (p13h) were hybridized to the DNA samples. The P3A an error of one part in four billion. Using a com- probe hybridizes to the 1.8kb DNA fragment in the homozygous normal (AA) and heterozygous individuals; similarly, the and it (Ap3th) bination of genetic gene cloning techniques, P3thprobe hybridizes to the same fragment of chromosomal DNA is possible to identify a person who is either hetero- from the heterozygous (AI3th) and 13-thalassemic individuals zygous or homozygous for the normal or mutant (f3thpth). The high molecular weight DNA hybridizing to the ra- dioactive probes represent sequences that are highly repeated in nucleotide substitution in the 3-globin gene. the genome, but contain partial complementarity to the synthet- The procedure is quite simple, a chromosomal ex- ic oligonucleotide probes. tract of an individual's cells is fragmented with re- striction endonucleases. The fragments are then sep- arated by gel electrophoresis, transferred to disorders will be detectable by using synthetic DNA nitrocellulose paper, and then incubated with a ra- probes coupled to either radioisotopic or fluorescent dioactively labeled oligonucleotide probe. If the tags. The widespread use of synthetic DNA probe is complementary to the normal gene, then hybridization technology for the detection of ge- it hybridizes to those fragments which contain the netic defects will result in a revolution in the health normal gene (Figure 4a); it will hybridize to a lesser care industry. We foresee a time when clinicians extent to fragments from a heterozygous individual. will use a battery of synthetic oligonucleotide tests However, the probe will not hybridize to any frag- to diagnose genetic diseases. Synthetic oligonucleo- ments from homozygotes without the normal gene. tide based tests will also be used to counsel couples The experiment can be reversed and done with a planning to have children. Thus, the probabilities probe that is complementary to the mutant se- of offspring inheriting certain genetic traits or de- quences (Figure 4b). The use of synthetic DNAs as diagnostic, clinical tools is in its infancy. In the near future, genetic

444 THE AMERICAN BIOLOGY TEACHER, VOLUME 46, NO. 8, NOVEMBER/DECEMBER 1984 Use of Synthetic DNA Under the appropriate conditions, the double for the Mutationof Genes stranded (ds) plasmid containing the cloned gene can be enzymatically degraded (Figure 5a) so that a Prior to recombinant DNA technology molecular single stranded (ss) plasmid molecule remains(Figure biologists and geneticists were confined to studying 5b). The synthetic oligonucleotide is then mixed gene function through the isolation of phe- with the ss circular DNA and the reaction mixture notypically selectable mutants. The classical tech- is heated. As the reaction mixture cools, the syn- niques of inducing mutations in genes included thetic oligonucleotide anneals (establishes base pair- chemical mutagens, ultraviolet light and ionizing ing) to the complementary region on the ss circular radiation. However, since the entire organism was DNA (Figure 5b). In the presence of all four deox- subjected to the mutagenesis regime, multiple muta- ynucleoside triphosphates the bacterial enzyme E. tions often arose. The desired mutation could then coli DNA polymerase elongates from the primer only be identified using complex genetic manipula- copying the nucleotides in the ss circulartemplate. tions. The in vitro mutagenesis of cloned genes al- This results in the synthesis of a heteroduplex mole- lows for the precise mutation (base substitution, de- cule (one nucleotide strand being derived from the letion or insertion) of the specific gene desired synthetic oligonucleotide, the other from the ss cir-

(Shortle, DiMaio, and Nathans 1981). cular molecule). Downloaded from http://online.ucpress.edu/abt/article-pdf/46/8/440/86223/4447895.pdf by guest on 01 October 2021 With the advent of molecular cloning meth- When it is desired to introduce a mutation into a odologies, biologists are now able to create muta- gene the same approach is used. A synthetic tions in both regulatory and structural genes in oligonucleotide containing the designed mutation vitro, transform the cloned gene back into the or- (mutator)is annealed to an ss template. However, ganism of interest, and study its biological conse- in this experiment the enzymatic reaction is carried quences. In some cases the mutation may not result out at a very low temperature (less than 10?C).The in a phenotypic alteration; on the other hand, lowered temperaturestabilizes base pair mismatches some mutations may have drastic, sometimes lethal, (i.e. G. mispaired to T), insertions and deletions. effects on the organism's phenotype. After a heteroduplex molecule is synthesized (one Synthetic DNA mediated site-specific in vitro mu- nucleotide strand derived from the ss circularDNA tagenesis offers a relatively low-cost, systematic ap- the other from the oligonucleotide containing mu- proach to studying the effects of specific mutations tation), the reaction mixture is used to transforman on cloned genes. The principle of synthetic DNA E. coli host. mediated mutagenesis is based upon the ability of a The resulting transformantsare screened using the synthetic DNA oligonucleotide to base pair wih technique previously described. In this situation the complementary sequences within a cloned gene. mutagenic oligonucleotide is radioactively labeled

DNase I or restriction A ~~~~~~endonuclease4 ~ A. Exonuclease1 + ethidiumbromide

Supercoiled DNA Nicked DNA Single stranded (Form I) (Form 1I) circular DNA Mutant DNA

DNApolymerase large fragment Introduce Bi. + oligonucleotide iinto bacteria DNA Ligase Original Single stranded Primer-template Heteroduplex DNA circular DNA intermediate

Figure 5. Generalized scheme for synthetic DNA mediated site- in the presence of DNA polymerase, DNA ligase, and all four specific in vitro mutagenesis. deoxynucleoside triphosphates. The newly synthesized hetero- 5a. Supercoiled DNA (plasmid) containing the cloned gene is duplex molecule (one nucleotide strand derived from the ss cir- digested enzymatically in two sequential reactions to produce a cular template, the other from the newly synthesized strand con- single stranded (ss) circular DNA molecule. taining the mutagenic oligonucleotide) is transformed into an E. 5b. The ss circular DNA molecule is mixed with the synthetic coli host. The plasmic containing the mutation can subsequently oligonucleotide bearing the desired mutation. Under conditions be isolated after one round of semi-conservative DNA replication that favor base pair mismatches, the synthetic oligonucleotide can in the host cell. be used to direct the synthesis of the complementary DNA strand

CHEMICAL SYNTHESIS OF DNA 445 and used to screen the transformants.Under appro- sequences coding for the initiation of mRNA tran- priate conditions of temperature and salt concentra- scription (promoters) are helping scientists under- tion, only those transformantscontaining the mutat- stand why some genes are more frequently tran- ed gene will hybridize to the probe. It should be scribed than others, and why certain tissue types pointed out the primary transformantswill contain express some genes but not others. a 50/50 mixture of nonmutant and mutant plasmids after the first round of semiconservative DNA rep- Concluding Remarks lication within the E. coli host. Therefore, it is sometimes necessary to isolate the plasmid DNA Since the development of solid phase methods from a "positive" clone and retransformE. coli host and commercial availability of , the chem- in order to obtain an E. coli transformant contain- ical synthesis of DNA oligonucleotides has become a ing only the mutant plasmid. The gene is ultimate- routine laboratory procedure. The increasing avail- ly sequenced to determine if the desired mutation ability of synthetic DNA sequences together with has been introduced. The gene then can be trans- the advent of molecular cloning techniques has had formed into the appropriate host in order to assay a profound effect on many biological studies. Syn- its effects in vivo. thetic DNA has become an indisposable tool in Synthetic DNA oligonucleotides have already modern biology laboratories. been used to create mutations in many genes. They Downloaded from http://online.ucpress.edu/abt/article-pdf/46/8/440/86223/4447895.pdf by guest on 01 October 2021 are currently being used commerciallyin the modi- Acknowledgements fication of industrially important proteins in an at- The authors wish to express their appreciation to Drs. tempt to alter their activities. For example, several K. Itakuraand R.B. Wallace for their helpful discussions, industrial labs are attempting to enhlance the cata- and MarleneNeerman for her expert technical assistance. Some of the work described in this report was supported lytic activity of enzymes used in commercially by the National Institutes of Health Grant GM30134and importantreactions by specifically altering the genes the National Science Foundation Grant PCM8108280to coding for these proteins. In vitromutations at DNA JohnJ. Rossi.

to the TLCwith slides of drying spinach leaves. We worked Letters Editor closely on many projects and she I read with much interest your ar- helped me demonstrate the TLC lab Eye-Oh ticle, "Thin Layer Chromatography lesson at meetings of biology teachers. (TLC) of Biology teaching has forsaken sci- Chlorophyll Pigments" in Unfortunately, Mrs. Hechtlinger's the April 1984 ence for pedagogy when issue of the American publisher did not design her hand- today's Biology Teacher. teacher, beset with demands for ac- Unfortunately, your book to include references or cita- search of the countability, associates the expression literature only went tions; as a consequence your study of back to IO (eye-oh) only with Instructional 1968, and you did not find her handbook did not disclose the or- my article of 21 igin Gone, it seems, are the years ago in the of my procedure, which she de- Objectives. same journal: teachers for whom this term would "Rapid Thin Layer scribed. conjure up the delicate wings of the Chromatographyof Chloroplast Pig- I note that many of your solutions ments for Io moth or the stately spire of the the High School Biology differ from those I employed, and Laboratory," genus Io in the molluscan family, AmericanBiology Teach- would not be surprised to learn that er, 25:8, Pleuroceratidae. Rare, today, is the December 1963. As far as I they work equally well, or better. know, mine teacher who can identify Io as the was the first paper to Being of a similar mind, we both describe a daughter of Inachus who, loved procedure for TLCof chlo- employed many of the same kinds of by roplast Zeus, was transformed into a white pigmentsusing microscopeslides. photographs. I heifer and later had a satellite of did not originate the idea of My procedure was used by many using Jupiter named after her. Who knows, microscope slides as chromato- schools in the metropolitan New or now even cares, that in 1907 Io plates. As described in my article, I York area, but has recently been was the symbol for ionium, an ele- obtained this idea from Peifer (1982), abandoned as part of the program to ment that lay between uranium and whose comprehensive paper I cite in avoid toxic chemicals in the high radium in the path of radioactive de- my article. school laboratory. One of your important citations is cay? David Kraus Old-timers admit that new educa- Hechtlinger's Handbookof modernex- periments for high school biology, 99 S. ParkAvenue, Apt. 214 tional priorities, reflecting the pres- RockvilleCentre, NY 11570 sures of a changing society, must (1971). Adelaide Hechtlinger (de- bring new meanings to our idiom. ceased) was my laboratoryassistant at the Far But never, they vow, shall ped- Rockaway High School in CORRECTION aguese-ical innovation transmogrify New York City and set out the mate- rials for class laboratorylessons in ac- In the September ABT Letter to the the cry of the Lone Ranger, "Heigh- Editor by Bill D. Davis, entitled "Re- oh, Silver!" cordance with my procedure. She later made the discovering TLC," the date of the issue significant contribu- in which he first read about the TLC David Kraus tion of using dehydrated leaves, pur- method was mistakenly printed as 1983. 99 S. Park Ave. Apt. 214 chased in the supermarket,instead of It should have read "1963." Rockville Centre, NY 11570 going through the tedious procedure

446 THE AMERICAN BIOLOGY TEACHER, VOLUME 46, NO. 8, NOVEMBER/DECEMBER 1984