- 1.MILESTONESM I L E S TO N E 1Keep em separated In the early 1950s, the biochemicalSmithies demonstrated that gels community started thoroughly made from starch solutions could investigating the chemical reactivitybe used to separate human serum of DNA and RNA, with the hopes proteins: when a hot starch solution that these studies would help to elu-was poured into a plastic tray, the cidate the molecular structures andcooled solution would form a solid cellular functions of these(but brittle) gel. Protein samples macromolecules.could be loaded into the gel, which One topic of interest was thewould then act as the stationary molecular mechanism of RNA phase, much like the Whatman paper and DNA hydrolysis; an important described above. Gel electrophoresis milestone in this field was publishedwas further refined, and, 12 years in 1952, when Markham and Smithlater, Loening demonstrated that gels reported that the hydrolysis of RNAmade from polymerized acrylamide proceeded via a cyclic phosphate and bisacrylamide (polyacrylamide intermediate, which was then further gels) had sufficient resolving power hydrolysed to produce a nucleoside to separate high-molecular-weight 2-monophosphate or 3-monophos- pieces of RNA. phate. A key development that led to Despite the broad utility of the this discovery involved the separation electrophoretic techniques, it was of complex mixtures of hydrolysedstill relatively difficult to separate RNA using a simple device, termedextremely large pieces of DNA for an electrophoresis apparatus.example, whole chromosomes The device was constructed fromfrom one another. In the mid-1980s, Whatman number 3 paper, severalSchwartz and Cantor described a new museum jars and various buffer approach, named pulse-field gradi- solutions; the hydrolysed ribonucleicent gel electrophoresis, which used acids were deposited onto the paper, short pulses from perpendicular and a power supply was attachedelectrical fields to separate large to the device. Applying the currentpieces of DNA. Pulse-field gel could have predicted that spotting led to the separation of the complex electrophoresis has since allowedhydrolysed ribonucleic acids on mixture into its components; biologists to undertake massiveWhatman paper would pave the way remarkably, relatively minor genotyping studies, as well as for the genomic era? These four differences in the structure of themolecular epidemiological analysesJoshua M. Finkelstein, molecules in the mixture led toof pathogens.Senior Editor, Naturearticles observable differences in mobility These four articles opened theopened across the paper. Using this approach, door to numerous other DNA tech- ORIGINAL RESEARCH PAPERS Markham, R. & the door to the authors were able to isolate nologies described in this collection;Smith, J. D. The structure of ribonucleic acids. I.Cyclic nucleotides produced by ribonuclease and numerous cyclic nucleotides, suggesting thatmany common molecular biology by alkaline hydrolysis. Biochem. J. 52, the hydrolysis of RNA proceeded viatechniques would not be possible552557 (1952) | Smithies, O. Zone electrophoresisother DNA the formation of a cyclic phosphateif there were not robust methodsin starch gels: group variations in the serum technologiesproteins of normal human adults. Biochem. J. 61, intermediate.for rapidly purifying and analysing 629641 (1955) | Loening, U. E. The fractionation ofdescribed This general approach was notnucleic acids of various sizes. It is high-molecular-weight ribonucleic acid bypolyacrylamide-gel electrophoresis. Biochem. J. in this restricted to small molecules hard to imagine a modern molecular102, 251257 (1967) | Schwartz, D. C. & Cantor, C. R. electrophoresis could also be per- biology laboratory that does notSeparation of yeast chromosome-sized DNAs bycollection... formed on larger biomolecules if sev-use some kind of electrophoreticpulsed field gradient gel electrophoresis. Cell 37,6775 (1984) eral adjustments were made. In 1955, technique on a regular basis. Who NATURE MILESTONES | DNA TECHNOLOGIES O CTOBER 2007 | S5 2007 Nature Publishing Group
2. MILESTONESbacterial plasmid. Using the restric- CORBIS M I L E S TO N E 2 tion enzyme EcoRI, they generatedThe dawn of recombinant DNA fragments from two plasmids (each conferring resistance to one antibiotic), joined them using DNA ligase and applied the mixture to The ability to make recombinant held by hydrogen-bond pairing in atransform E. coli. As they had hoped, DNA molecules is the cornerstonedouble-stranded configuration.a fraction of the transformed bacteria of modern molecular biology.DNA ligase, which was the first became resistant to both antibiotics Yet 40 years ago, it was hardly a ingredient for making recombinant while carrying a single hybrid conceivable accomplishment. DNA, was then at hand, butplasmid. Not only had they demon- In the 1960s, biologists hadother ingredients, like restric-strated that bacterial plasmids con- realized that DNA recombination tion enzymes, (see Milestone 4) structed in vitro were functional in happens in the cell for example,remained to be discovered. Anotherbacteria, but they had also described when breaks caused by ultraviolet key concept was the use of plasmids the first plasmid vector. irradiation are repaired and theas vectors for shuttling DNA into Meanwhile, Paul Berg had devised search for an enzyme that could bacteria. Stanley Cohen, who wasa similar experiment to transfer join DNA molecules was on. Thestudying the role of plasmids inforeign DNA into mammalian cells, breakthrough came at the beginningbacterial resistance to antibiotics using the tumour virus SV40 as a of 1967, when Martin Gellert at the at Stanford University, first workedvector. In 1972, he made a hybrid National Institutes of Health showedout a transformation method tomolecule in vitro by inserting that Escherichia coli extracts couldmake bacteria take up purifiedphage sequences into SV40. These convert phage DNA hydrogen-plasmid DNA.reports immediately raised concerns, bonded circles into a covalently Then, in 1973, Cohen and hisas E. coli, which is a natural habitant circular form. Within 6 months, Stanford colleague Annie Chang, inof the human gut, could now carry Gellert and three other groupscollaboration with Herbert Boyer andhybrid DNA molecules containing independently purified the enzymaticRobert Helling at the University of SV40 oncogenes or other potentially activity, which formed phospho- California in San Francisco, reported harmful sequences. These fears led diester bonds between DNA endsthe first in vitro construction of athe community to a self-imposedM I L E S TO N E 3 and in an easily identifiable region of thecell. They were able to prepare tritiatedcomplementary RNA of adequate purityFully cooked FISHand activity to determine the conditionspermitting clear visualization ofthe extrachromosomally amplifiedribosomal DNA. They predicted that Fluorescence in situ hybridization (FISH) easy testing and validation of the labelling the RNA at a higher specificity has become a methodological mainstaymethods was also needed. would permit the detection of non- in many fields. It permits the detection, In the 1960s, Joe Gall and Mary Louduplicated genes in chromosomes. quantification and localization ofPardue at Yale University were studyingPardue later went to work with genes and RNA at resolutions rangingthe extrachromosomal amplification ofmembers of the Max Birnstiel laboratory. from single nucleotides to wholeribosomal RNA genes in Xenopus laevis. Using Drosophila melanogaster polytene chromosomes, or even whole cells, using This system conveniently provides achromosomes, they could visualize a fluorescently labelled complementaryknown sequence at a high copy number the location of histone genes on DNA or RNA probe. Remarkably, whileindividual chromosomes. However, the immunofluorescence detection ofuse of radioactivity, high background protein has been around since 1949, andlevels and long exposure times were immunofluorescent stains for nucleic problematic, and multiplexing was acids soon followed, FISH was not fully impossible. It was not long before cooked until the early 1980s. fluorescently labelled nucleic acids The principal development that led provided the final crucial tool that gave to FISH as we know it today was theus the sensitive multiplexing versions of determination of the sample fixation FISH we have today. and permeabilization conditionsTwo different methods for fluorescently necessary to fix cells in their native labelling nucleic acids are commonly structure, while allowing theused for FISH. The direct method, first introduction and specific annealing of described by P. van Duijn and colleagues, complementary labelled nucleic-acidrelies on direct labelling of the nucleic molecules. A system that would permitacid with fluorophores and was the first S6 | O CTOBER 2007 www.nature.com/milestones/dnatechnologies 2007 Nature Publishing Group 3. moratorium on recombinant DNAM I L E S TO N E 4 experiments. However, the founda- tion had been laid and progress soon resumed.Veronique Kiermer, Chief Editor,Making the cutNature MethodsI remember clearly the first time I made a supervised ORIGINAL RESEARCH PAPERS Gellert, M. trip, as an undergraduate student, to the departmental Formation of covalent circles of DNA by E. coli extracts. Proc. Natl Acad. Sci. USA 57, 148155freezer to obtain a precious aliquot of restriction enzyme. (1967) | Cohen, S. N., Chang, A. C. Y. & Hsu, L. Its real value, however, only dawned on me when I created Nonchromosomal antibiotic resistance inthe first of countless recombinant DNA constructs. bacteria: genetic transformation of E. coli by R-factor DNA. Proc. Natl Acad. Sci. USA 69,It is unlikely that Stuart Linn and Werner Arber were 21102114 (1972) | Cohen, S. N., Chang, A. C. Y.,aware of the Boyer, H. W. & Helling, R. B. Construction offar-reaching c
