A Evolução da Eletroforese e suas Contribuições para a Biologia Molecular

In the early 1950s, the b iochemical comm unity started thoro ughly inv estigatin g the chemical r eactivity of DN A and RN A, with the hopes that these studies would hel p to elu- cidate the mo lecular structures an d cellula r function s of these macrom olecules. One top ic of int erest was the molecular mecha nism of RN A and DN A hy drolysis; a n importan t milestone in this field was published in 1952, when Markham a nd Smith reported tha t t he h ydrol ysis of RN A pr oceeded via a cyclic phospha te inte rmediate , which was then further hy drol ysed to produce a n ucleoside 2 ′ -monopho sphate or 3 ′ -monophos- pha te. A key develop ment tha t led to this discovery invo lved the separa tion of com plex mixtures o f hy drolysed RN A using a simple device, termed an ‘ electrop hor esis appara tus ’ . The device was constructed from Wha t man n umber 3 paper , se veral museum jar s and vario us buffer solutio ns; the hy drolysed ribon ucleic acids were deposit ed ont o the paper , and a pow er suppl y was attach ed to the device. A pplying the curren t led to the separa tion of the com plex mixture in to its com ponents; remarkably , relatively m inor differences in the structure o f the molecules in the mixture led to obser vable differences in mob ility across the pa per . U sing this app roach, the au thors wer e able to isola te ‘ c yclic ’ nucleotides, suggestin g that the hy drol ysis of RN A proceeded via the forma tion of a cyclic ph osphat e intermed i ate. This general ap proach was not restricted to small mo lecules — ‘ electropho resis ’ could also be per- formed o n larger bio molecules if sev- eral adjustmen ts were made. In 1955, Smithies demon strated tha t gels made from star ch solutio ns could be used to separa te huma n serum pro teins: when a hot starch sol ution was poured in to a plastic tray , the cooled solu tion would f orm a solid (but b rittle) ‘ gel’ . Pro tein samp les could be loaded int o the gel, which would then act as the stationary phase, m uch like the W hatman pa per described above. Gel electro phor esis was further refined, and, 12 years later , Lo ening demon strated tha t gels made from polymerized acry lamide and bi sacr ylamide (‘pol yacr ylamide gels ’) had suff icient r esolving power to separa te high-molecular -weight pieces of RN A. Despit e the broad u tility of the electrop horetic t echniques, it was still rela tively difficult to separa te extremely la rge p ieces of DN A — for exam ple, whole chr omosomes — from on e another . In the mid-1980s, Schwartz and Can tor described a ne w ap proach, na med ‘pulse-field gradi- ent g el electrop horesis, ’ w hich used short pulses from perpendic ular electrical field s to sepa rate larg e pieces of D NA. Pulse-field gel electrop horesis has since allo wed biologists to undertake massive genotypin g studies, as well as molecular epidemio logical analyses of pa thogens. These four articles o pened the door to numer ous other DN A tech- nologies described in this collection; man y common mo lecular biology techniques wo uld not be p ossible if there wer e not rob ust met hods for ra pidly purifying and a nalysing nucleic acids o f various sizes. I t is hard t o imagine a modern molecular biology laborat ory that does not use som e kind of el ectro pho retic technique o n a regular basis. W ho could ha ve pr edicted that spotting hy drolysed ribon ucleic acids on Wha t man pa per would pa ve the way for the geno mic era? Joshua M. Finkelste in, Senior Editor , Natu re ORIGINAL RESEARCH P APERS Markham, R. & Smith, J. D. The structure of ribonucleic acids. I. Cyclic nucleotides produced by ribonuclease and by alkaline hydrolysis. Biochem. J. 52 , 552–557 (1952) | Smithies, O. Zone electrophoresis in starch gels: group variations in the serum proteins of normal human adults. Biochem. J. 61 , 629–641 (1955) | Loening, U. E. The fractionation of high-molecular -weight ribonucleic acid by polyacrylamide-gel electrophoresis. Biochem. J. 102 , 251–257 (1967) | Schwartz, D. C. & Cantor , C. R. Separation of yeast chromosome-sized DNAs by pulsed field gradient gel electrophor esis. Cell 37 , 67–75 (1984) MILEST ONE 1 K eep ‘em separ ate d These four articles opened the door to numerous other DNA technologies described in this collection... NA TURE MILEST ONES | DNA TECHNOL OGIES O CTOBER 2007 | S5 MILEST ONES © 2007 N a ture Publi s hi n g G r o u p The abili ty to make r ecomb inant DN A molecules is the cornerstone of modern molecular biology . Y et 40 years ago , it was hardly a conceivab le accom plishment. In the 1960s, bio logists had realized tha t DN A recomb ination hap pens in the cell — for exam ple, when breaks ca us ed by ul traviolet irradiation a re r epaired — a nd the search f or an enzyme tha t could join DN A molecules was on. The break through came a t the beginning of 1967, when M artin Gellert at the N atio nal In stit utes o f Health sh ow ed tha t Esc herich ia col i extracts could con vert λ phag e DN A ‘hydrogen- bonded circles ’ int o a covalen t ly circular fo rm. W ithin 6 mon ths, Gellert and three other gr oups independently purified the enzymatic activity , which formed ph ospho- diester bonds between DN A ends held by h ydrogen-bond pairing in a double-stran ded configuration. DN A ligase, which was the f irst ingredien t for making reco mbinant DN A, was then at hand, bu t other ingr edients, like r estric- tion enzymes, (see Milestone 4) remained t o be discovered. Ano ther key concept was the use of plasmids as vectors fo r shu ttling DN A into bacteria. Stanley Cohen, who was studying the r ole of plasmids in bacterial resistance to a ntibio t ics at S t anfo rd U niversi ty , f irst wo rked out a ‘ transfo rmation ’ method to make ba cte ri a t ake up pur if ie d plasmid DN A. Then, in 1973, Cohen and his Stanf ord co lleague Annie Chang, in collabora tion with Herbert Boyer an d Robert Helling a t t he U niversity o f California in San F rancisco , reported the fi rst in v itro construction of a MILEST ONE 2 The dawn of r ecombinant DNA Fluorescence in situ hybridization (FISH) has become a methodological mainstay in many fields. It permits the detection, quantification and localization of genes and RNA at resolutions r anging from single nucleotides to whole chromosomes, or even whole cells, using a fluorescently labelled complementary DNA or RNA probe. Remarkably, while immunofluorescence detection of protein has been ar ound since 1949, and immunofluorescent stains for nucleic acids soon followed, FISH was not ‘fully cooked’ until the early 1980s. The principal development that led to FISH as we know it today was the determination of the sample fixation and permeabilization conditions necessary to fix cells in their native structure, while allowing the introduction and specific annealing of complementary labelled nucleic-acid molecules. A system that would permit easy testing and validation of the methods was also needed. In the 1960s, Joe Gall and Mary Lou P ardue at Y ale University were studying the extrachr omosomal amplification of ribosomal RNA genes in Xenopus laevis . This system conveniently provides a known sequence at a high copy number MILEST ONE 3 F ully cooked FISH and in an easily identifiable region of the cell. They were able to pr epare tritiated complementary RNA of adequate purity and activity to determine the conditions permitting clear visualization of the extrachr omosomally amplified ribosomal DNA. They predicted that labelling the RNA at a higher specificity would permit the detection of non- duplicated genes in chromosomes. P ardue later went to work with members of the Max Birnstiel laboratory . Using Drosophila melanogaster polytene chromosomes, they could visualize the location of histone genes on individual chromosomes. However , the use of radioactivity, high backgr ound levels and long exposure times wer e problematic, and multiplexing was impossible. It was not long before fluorescently labelled nucleic acids provided the final crucial tool that gave us the sensitive multiplexing versions of FISH we have today . T wo different methods for fluorescently labelling nucleic acids are commonly used for FISH. The ‘direct’ method, first described by P . van Duijn and colleagues, relies on dir ect labelling of the nucleic acid with fluorophor es and was the first bacterial plasmid. U sing the restric- tion enzyme EcoRI , they genera ted fragments from tw o plasmids (each conferring r esistance to one an tibiotic), joined them using D N A ligase and ap plied the mixtur e to transf orm E. co li . As they had hoped, a fraction of the tran sformed bacteria became resistan t to b oth an tibiotics while carr ying a single h y brid plasmid. N ot only had they demon- strated tha t bac terial plasmids con- structed in v itro wer e functional in bacteria, but they had also described the first plasmid vector . M ean while, P aul Berg had devi sed a similar experiment to transfer for eign DN A into mammalian cells, using the tumour virus SV40 as a vector . In 1972, he made a hybrid molecule in vit ro b y inser ting λ phage sequences in to SV40. These reports immedia tely ra ised concerns, as E. col i , w hich is a nat ural habi tant of the h uman gut, could no w carr y hybrid DN A molecules conta ining SV40 oncogenes o r other potentially harmful sequences. These fears led the comm unity to a self-im p osed CORBIS MILEST ONES S6 | OCTOBER 2007 ww w.natur e.com/ milestones/ dnatechnologies © 2007 N a ture Publi s hi n g G r o u p implementation of FISH. The ‘indirect’ method, developed later by the David W ard gr oup and implemented in commonly used FISH kits, employs immunogenic or enzymatic detection of tagged nucleic-acid probes following hybridization. As improvements to the ‘r ecipe’ continue to be made, FISH is finding its way onto the plate of increasing numbers of resear chers and promises to do so for the foreseeable futur e. Daniel Evanko, Senior Editor , Nature Methods ORIGINAL RESEARCH P APERS Gall, J. G. & P ardue, M. L. Formation and detection of RNA–DNA hybrid molecules in cytological preparations. Proc. Natl Acad. Sci. USA 63 , 378–383 (1969) | P ardue, M. L., Kedes, L. H., W einberg, E. S. & Birnstiel, M. L. Localization of sequences coding for histone messenger RNA in the chromosomes of Drosophila melanogaster . Chr omosoma 25 , 135–151 (1977) | Bauman, J. G., Wiegant, J., Borst, P . & van Duijn, P . A new method for fluorescence microscopical localization of specific DNA sequences by in situ hybridization of fluorochrome labelled RNA. Exp. Cell. Res. 128 , 485–490 (1980) | Langer , P . R., W aldrop, A. A. & W ard, D. C. Enzymatic synthesis of biotin- labeled polynucleotides: novel nucleic acid affinity probes. Proc. Natl Acad. Sci. USA 78 , 6633–6637 (1981) mora torium o n recom binan t DN A experiments. H owever , the founda- tion had been laid and p rogress soon resu m e d . V eroni que K ier mer , Chief Ed ito r , Natu re Me t h o ds ORIGINAL RESEARCH P APERS Gellert, M. Formation of covalent cir cles of λ DNA by E. coli extracts. Proc. Natl Acad. Sci. USA 57 , 148–155 (1967) | Cohen, S. N., Chang, A. C. Y . & Hsu, L. Nonchromosomal antibiotic resistance in bacteria: genetic transformation of E. coli by R-factor DNA. Proc. Natl Acad. Sci. USA 69 , 2110–2114 (1972) | Cohen, S. N., Chang, A. C. Y ., Boyer , H. W . & Helling, R. B. Construction of biologically functional bacterial plasmids in vitro . Proc. Natl A cad. Sci. USA 70 , 3240–3244 (1973) | Jackson, D. A., Symons, R. H. & Berg, P . Biochemical method for inserting new genetic information into DNA of simian virus 40. Proc. Natl Acad. Sci. USA 69 , 2904–2909 (1972) FUR THER READING Gefter , M. L., Becker , A. & Hurwitz J. The enzymatic repair of DNA, I. Formation of cir cular λ DNA. Proc. Natl Acad. Sci. USA 58 , 240–247 (1967) | Oliver a, B. M. & Lehman, I. R. Linkage of polynucleotides through phosphodiester bonds by an enzyme from E. coli . Proc. Natl Acad. Sci. USA 57 , 1426–1433 (1967) | W eiss, B. & Richardson, C. C. Enzymatic breakage and joining of deoxyribonucelic acid. Proc. Natl Acad. Sci. USA 57 , 1021–1028 (1967) | Zimmerman, S. B., Little, J. W ., Oshinsky, C. K. & Gellert, M. Enzymatic joining of DNA strands: a novel reaction of diphosphopyridine nucleotide. Proc. Natl Acad. Sci. USA 57 , 1841–1848 (1967) I remember clearly the first time I made a supervised trip, as an undergr aduate student, to the departmental freezer to obtain a pr ecious aliquot of restriction enzyme. Its real value, however , only dawned on me when I created the first of countless recombinant DNA constructs. It is unlikely that Stuart Linn and W erner Arber were aware of the far -reaching consequences of their discovery when they stumbled across r estriction enzymes in the late 1960s. While studying a phenomenon called host-controlled restriction of bacteriophage gr owth, they showed that restriction enzymes of the host cells cleave unmethylated phage DNA in numerous places, thereby limiting their gr owth. A couple of years later , Hamilton Smith and Kent Wilcox reported the isolation and characterization of the first restriction enzyme — endonuclease R (later renamed Hin dII) — fr om extracts of Haemophilus influenzae strain Rd. Importantly, the enzyme degraded for eign DNA, such as that of phage T7, but did not affect native H. influenzae DNA. Smith and Wilcox demonstrated that endonuclease R pr oduces double- stranded 3 ′ -hydr oxyl, 5 ′ -phosphoryl cleavage products. They proposed that the enzyme recognizes a specific sequence on the for eign DNA, and estimated from the number of br eaks that the site would have to be five or six bases in length. Smith, together with Thomas Kelly, determined the recognition sequence using end-labelling techniques. This was an exceptional technical feat, as there was no method at the time for the analysis of terminal sequences beyond the dinucleotide level. They postulated that the internal symmetry of the recognition sequence, which was cleaved in the middle, was not surprising given that the enzyme carries out a symmetrical reaction on opposite str ands. Before long, Kathleen Danna and Daniel Nathans pioneer ed the application of restriction enzymes. They used endonuclease R to char acterize the small oncogenic DNA virus SV40: the resulting 11 fr agments were r esolved by polyacrylamide gel electrophor esis, and their molecular weights were determined. Their prediction that r estriction-enzyme analysis would be useful to map a genome region and to localize specific genes by testing for biological activity turned out to be visionary . The ‘recombination’ potential of r estriction enzymes was first demonstrated by Janet Mertz and Ronald Davis. They showed that the R1 restriction endonuclease produces ‘stagger ed’ breaks, gener ating ‘cohesive’ ends that are identical and complementary . Their findings suggested that any R1-generated ends can be joined by incubation with DNA ligase to generate hybrid DNA molecules. Thus, the era of r ecombinant DNA technology was born. Arianne Heinrichs, Chief Editor , Nature Reviews Molecular Cell Biology ORIGINAL RESEARCH P APERS Smith, H. O. & Wilcox, K. W . A restriction enzyme from Hemophilus influenzae . I. Purification and general properties. J. Mol. Biol. 51 , 379–391 (1970) | Kelly, T . J. Jr & Smith, H.O. A restriction enzyme fr om Hemophilus influenzae . II. Base sequence of the recognition site. J. Mol. Biol. 51 , 393–409 (1970) | Danna, K. & Nathans, D. Specific cleavage of simian virus 40 DNA by restriction endonuclease of Hemophilus influenzae . Proc. Natl Acad. Sci. USA 68 , 2913 –2917 (1971) FUR THER READING Linn, S. & Arber , W . Host specificity of DNA produced by Escherichia coli , X. In vitro restriction of phage fd replicative form. Pr oc. Natl. Acad. Sci. USA 59 , 1300–1306 (1968) | Mertz, J. E. & Davis, R. W . Cleavage of DNA by R1 restriction endonuclease generates cohesive ends. Proc. Natl Acad. Sci. USA 69 , 3370–3374 (1972) MILEST ONE 4 Making the cut Remarkably , while immuno- fluorescence detection of protein has been around since 1949… FISH was not ‘fully cooked’ until the early 1980s . Stockbyte NA TURE MILEST ONES | DNA TECHNOL OGIES O CTOBER 2007 | S7 © 2007 N a ture Publi s hi n g G r o u p The central dogma of mo lecular biology states that DN A makes RN A makes protein, y et one o f the most important D N A technologies stemmed from the discovery that the first of these steps can be reve rs e d. In 1970, p uzzled by the ab ility of RN A tumour viruses to stably transfo r m cells — withou t incorpora- tion of a DN A copy of viral g enes into the host geno me — Baltimore, and T emin and Mizutani , looked for DN A polymerase activity in puri- fied prepa ration s of such viruses. The kinetics of the incorpora tion of radiolabelled th ymine indicated tha t DN A was being synthesized. The reaction was sensitiv e to ribonuc lease trea tment, sho wing that it was RN A-dependen t, whereas the prod uct was deoxyribonuclease- sensitive b ut ribon uclease-insensiti ve , confirming that i t was DN A. T wo years later , it was demon- strated tha t the reverse transcrip tase could be used in v itro to synthesize cDN A from mammalian mRN As. Ve r m a et al. and K acian et al. bo th added prepa ration s of globin mRN As to revers e tr anscr iptas e from avi an my eloblastosis virus. They correctly hypothesized tha t the reaction would o nly work efficiently if they also added oligo(dT), which wo uld hybridize t o the poly(A) tail of the mRN As and act as a primer . By hybridizing their D N A product to the original mRN A templa te, they confirmed that they had successfully synthesized cDN A. Over the next two decades, reverse tran scriptase was widely used for the clo ning of expressed genes. Ho wever , one of the biggest t echnical hur dles, especially for the cr eation of com prehensive lib raries, was that most cDN As in a given r e action wer e not full length owing t o prema ture termination. I n addition, low-ab un- dance transcrip ts were fa r less likely to be cloned tha n hig h-abunda nce ones. I n the lat e 1990s, Carninci, MILEST ONE 5 A r ev erse pr o v es to be an adv ance It was a simple but clever idea that, in 1975, led Edwin Southern to invent a method that carries his name and that has revolutionized the study of DNA. In the early 1960s, Julius Marmur and P aul Doty published a study that accurately described the conditions for the optimal renatur ation of DNA complementary strands, upon denaturation by high temper atures. They proposed that high temper ature was requir ed to block the formation of weak bonds between non- complementary strands and to guarantee the pr oper pairing of complementary molecules. Given these findings and the intrinsic features of DNA molecules, it became evident that a specific DNA fragment could be identified from a biological sample by letting it hybridize, following denaturation, to a r adiolabelled complementary molecule. An RNA strand, for example, could be used as a probe to gain insight into gene structur e and function. The hybridization could even occur when the DNA was trapped onto a nitrocellulose membr ane. At that time, DNA fragments could be obtained by digestion with the recently discovered r estriction enzymes (see Milestone 4) and by separation thr ough MILEST ONE 6 Southern migr ation electrophor esis in agarose gel (see Milestone 1). However , DNA recovery from the gel inevitably led to loss of material and a notable decrease in the resolving power of electr ophoresis. Southern had a genial intuition that he could transfer the DNA fragments dir ectly from the gel onto a nitrocellulose membr ane laid on top of it. The agarose gel being permeable, he could force liquid to pass thr ough the gel by piling up, on top of the nitrocellulose, a stack of dry filter paper . Drawn by the flowing liquid, the DNA fragments could be soaked out of the gel. Following hybridization with radiolabelled RNA, autor adiography of the membrane r evealed the specific fragments containing the sequence of interest, which appear ed as sharp bands in the same position as they had been on the gel. It was finally possible to detect a specific DNA sequence from a smear , without having to purify it away from the rest of the genome. Southern used his method to study bacterial and eukaryotic ribosomal genes, but its practice soon became widespread. It facilitated the study Ha yashizaki and colleagues devel- oped several techniques to over come these problem s. By bio t in cap ping o f the mRN As, they ensured tha t only full-length cDN As were selected. After first- strand cDN A synthesis, RN Ase I was used to destro y an y part of an y mRN A t hat was no t bound to cDN A. This caused the remo val of the 5 ′ bio tin cap fr om the mRN As of all non-com plete cDN As. Magnetic be ads were u se d to s el ec t only th e full-length cDN As for second-strand synthesis and c loning. Their discovery that trehalose makes reverse tra nscriptase more thermostable meant that the r eac- tion could be ca rried out a t a higher tem peratur e, at whic h the forma t ion of fewer RN A secondary str uctures increased the num ber of full-length cDN As pr oduced. Finally , in or der to com plete expressio n libraries, they s electively cloned ra re new cDN As by screenin g out a bundan t ones and those already cloned in existin g libraries. T o achieve this, they added biotin ylated RN A f rom the o r iginal samp le and existing libra ries after first-strand MILEST ONES S8 | OCTOBER 2007 ww w.natur e.com/ milestones/ dnatechnologies © 2007 N a ture Publi s hi n g G r o u p of gene structures and, only a few years later , the discovery of genetic defects (such as the loss of a restriction enzyme site underling sickle cell anaemia). Nowadays, the applications of the Southern blot span from basic to biomedical resear ch, and from genetic engineering to forensics, yet the protocol r emains surprisingly similar to that described by Southern over 30 years ago. The method also inspired others to adopt a similar strategy for the study of different molecules. T o emphasize the similarity with the Southern blot, the transfer of RNA and pr otein from a gel to a solid support wer e named northern and western blot, respectively . Francesca P entimalli, Assistant Editor , Nature Reviews Cancer and Natur e Reviews Genetics ORIGINAL RESEARCH P APERS Marmur , J. & Doty, P . Thermal renatur ation of deoxyribonucleic acids. J. Mol. Biol. 3 , 585–594 (1961) | Southern, E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98 , 503–517 (1975) synthesis. All of the cD N A t hat hybridized to the RN A was remov ed with magnetic beads, leaving rar e cDN As behind. Fr om its o rigin as an esoteric pro p erty of certain viruses, reverse transcriptio n has become huge ly importan t in molecular biology . I ts influence extends from clo ning to the develop ment o f microarrays to the annotation of genomes. Pa trick Goymer , Associate Ed itor , Na ture Reviews Genetics ORIGINAL RESEARCH P APERS Baltimore, D. RNA-dependent DNA polymerase in virions of RNA tumour viruses. Nature 226 , 1209–1211 (1970) | T emin, H. M. & Mizutani, S. RNA- dependent DNA polymerase in virions of Rous sarcoma virus. Nature 226 , 1211–1213 (1970) | V erma, I. M. et al . In vitro synthesis of DNA complementary to rabbit reticulocyte 10S RNA. Nature New Biol . 235 , 163–167 (1972) | Kacian, D. L. et al . In vitr o synthesis of DNA components of human genes for globins. Nature New Biol . 235 , 167–169 (1972) | Carninci, P . et al . High-efficiency full-length cDNA cloning by biotinylated CAP trapper . Genomics 37 , 327–336 (1996) | Carninci, P . et al . Thermostabilization and thermoactivation of thermolabile enzymes by trehalose and its application for the synthesis of full length cDNA. Proc. Natl Acad. Sci. USA 95 , 520–524 (1998) | Carninci, P . et al . Normalization and subtraction of cap-trapper -selected cDNAs to prepare full- length cDNA libraries for rapid discovery of new genes. Genome Res . 10 , 1617–1630 (2000) A fundamental corner stone o f the era of “ genetic engineering ” was the developmen t of technology that allo wed researchers t o determine a DN A sequence in its linear or der . Given the adven t of electropho resis (see Milestone 1), the question s became how could o ne genera te fragments a t ever y position an d how could the terminal base of the fragments be distinguished? Three methods were r e volu t ionary in achiev- ing these goals. The first, cleavin g single-stranded DN A (ssDN A) or denatured do uble-stranded DN A (dsDN A) labelled at one end, was pu blished by M axam and G ilber t in 1977. Fragmen ts were generat ed in two steps: the base was removed, and then the weakened suga r bond was bro ken by the addi tion of alkali o r amines. In this chemi- cal method, the four bases wer e not specif ically determined; rather , one of the four r e actions cleaved a t pyrimidines, one cleaved a t C, and the other two had a pr eference for clea ving G > A or A > G. The four pools of fragmen ts were separat ed in differen t lanes of a polyacrylamide gel and the sequence, wi t h a read length of ~100 bases, was deduced from the ladder o f bands. Later tha t year , Sanger and co lleagues published a n enzymatic sequencing p rot ocol. This method did not inv olve D N A breakage, but exp loit ed the fact that dideoxy-n ucleotides (ddNTP s), lacking a 3 ′ -h ydro xy l, cannot be extended by D N A polymerase. Consequentl y , one co uld set up four D N A synthesis reactions con taining the same ssDN A templa te and primer , DN A polymerase, a mixture of the deo xynucle- otide (dNTP) and ddNTP f orms of o ne of the nucleo tides, and the remaining three dNTP s (one of which was labelled). A s b oth the ddNTP and dNTP were p resent, D N A p olymerase would sometimes incorpora te the correct dNTP , allo w- ing further polymerization, and a t other times would inco rporate the ddNTP , causing chai n termination. After D N A denaturatio n and poly- acr ylamide gel electro phoresis, a series of labelled bands corr esponding to termina tion at a specific nuc leotide could be r ead. I t was appr eciated tha t in order t o sequence genomes, i t would be necessar y to au toma te sequencing and ga t her informa tion in real time. In to this void st epped the Hood labora tory with a third technique , a variation o f the Sanger method that bypassed the ra te-limiting st ep — detection o f the labelled bands b y au toradi- ograp hy . Rather than using labelled dNTP s, the primer oligo nucleotides in the fo ur reactions were a ttached to fluor opho res with differen t emission maxima. The fo ur reactions yielded fragments tagged with differ ent fluor opho res, so that they could be co mbined a nd run in a single column g el. Sim u ltaneo usly , the group deve loped a detection system tha t us ed a laser to read the fluoro phore signals, an d an anal ytical progra m that r esolved the ra w data into a series of peaks that co rresponded to the sequence. When a sin gle lane con tained all four reaction s, 200 bases were read with ease. S ubsequentl y the fluoro phor es were a ttached to ddNTP terminato rs, remo ving the need fo r tagged primers a nd allowing all four r e actions to be performed in o ne reaction, slab gels w ere r eplaced with ca pillaries an d read lengths wer e extended to 600 or mo re bases. These app roaches facili tated the explosio n of sequence-gathering st udies that ha ve evolved in to our curren t bioinf ormatics-driv en research. Angela K. Eg gleston, Senior Editor , Natu re ORIGINAL RESEARCH P APERS Maxam, A. M. & Gilbert, W . A new method for sequencing DNA. Proc. Natl Acad. Sci. USA 74 , 560–564 (1977) | Sanger , F ., Nicklen, S. & Coulson, A. R. DNA sequencing with chain-terminating inhibitors. Proc. Natl Acad. Sci. USA 74 , 5463–5467 (1977) | Smith, L. M. et al. Fluor escence detection in automated DNA sequence analysis. Nature 321 , 674–679 (1986) WEB SITE DNA sequencing from Wikipedia: http://en.wikipedia.org/wiki/DNA_sequencing MILEST ONE 7 Deciphering the code MILEST ONES NA TURE MILEST ONES | DNA TECHNOL OGIES O CTOBER 2007 | S9 © 2007 N a ture Publi s hi n g G r o u p CORBIS Although the existence of variable loci in human DNA had been appreciated for some time, it was not until the late 1970s and early 1980s that a way to use polymorphisms for large- scale systematic mapping of human genes was proposed. David Botstein, Raymond White, Mark Skolnick and Ronald Davis argued that a lar ge number of DNA sequence polymorphisms must exist in the human population, and that some of these should be detectable as variants in the length of DNA fragments pr oduced by restriction enzymes (r estriction fragment-length polymorphisms or RFLPs). These RFLPs could be detected using Southern blotting experiments on human genomic DNA. Importantly, and unlike classical polymorphic antigenic and enzyme markers, these new loci could be identified in non-coding regions of the genome as well as within genes. Linkage relationships among RFLPs could be established using pedigrees, and genetic linkage to a locus of inter est would allow a gene to be mapped and defined, even if the RFLPs were not in the gene. Botstein and colleagues estimated that at least 150 highly polymorphic regions at r egular intervals in the human genome would make it feasible to construct a human genetic-linkage map and to localize disease genes. The first practical demonstr ation of this came in 1983 with the mapping of the gene for Huntington disease. As proposed by Botstein and colleagues, a large number of r ecombinant DNA probes defining RFLPs in human DNA had by then been identified. In a collaborative effort involving r esearchers in the United States and V enezuela, James Gusella and colleagues screened the DNA of members of two Huntington families with 12 such probes, and identified one that defined a marker on chromosome 4 with close linkage to the disease locus. A new field, positional cloning of genetic disease loci, was born. RFLPs were later involved in the mapping and positional cloning of the cystic fibrosis gene by John Rior dan and colleagues. Early linkage studies with RFLPs also allowed the creation of the first genome-wide genetic maps, which, together with sequence-tagged sites introduced by Maynar d Olson and colleagues, allowed the construction of sequence- based physical maps of genomes. Natalie de Souza, Associate Editor , Nature Methods ORIGINAL RESEARCH P APERS Botstein, D., White, R. L., Skolnick, M. & Davis, R. W . Construction of a genetic linkage map in man using restriction fragment length polymorphisms. Am. J. Hum. Genet. 32 , 314–331 (1980) | Gusella, J. F . et al. A polymorphic DNA marker genetically linked to Huntington’ s disease. Nature 306 , 234–238 (1983) FUR THER READING Riordan, J. R. et al. Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA. Science 245 , 1066–1073 (1989) | Olson, M., Hood, L., Cantor , C. & Botstein, D. A common language for physical mapping of the human genome. Science 245 , 1434–1435 (1989) MILEST ONE 8 Get out the map In the post-genomic era, understand- ing biological p rocess es increasingly relies o n analysis of g ene functions. Y et, until a few decades ago , studying eukaryotic gene function in v ivo was impossible, as no efficient and r epro- ducible p rocedur es to tran sfer DN A into eukaryotic cells were a vailable. In the la te 1970s, Gerald Fink and colleagues set the basis for stud ying eukaryotic genes by establishing a transf ormatio n protocol t o intr oduce exogenous D N A into yeast cells permanentl y . A few year s later , Mario Capecchi show ed that micr oin jection of the herpes simplex virus gene encoding thymidine kinase int o the nuclei o f mammalian cells lacking this enzyme allowed the kinase activity t o be reco ver ed. I t was only in 1982, however , that DN A was successfully manipula ted in v ivo in a hig her organism. Alla n Spradlin g and Gerald Ru bin charac- terized P -elements — mobile DN A elements — thro ug h analysis o f Droso phi la mela nogaster stra ins that gave rise to p rogen y suf fering from the hybrid dysgenesis g enetic syndrome. They identified two gr oups o f P -elements that differ ed in size and abili ty to move within the geno me. In jecting 3-kb au tonomo us P -elements into Dros oph ila embryos lacking them revealed tha t the elements could insert into ra ndom geno mic positions, ind ucing m utatio ns in a fraction of the pr ogeny . These find- ings initia ted the us e of P -elements in large-scale m utagenesis screens in Dro soph ila , given the adva ntage o f gene clonin g in the identified mu t ants. In mice, si te-directed mu t agenesis was first used in 1987, when two MILEST ONE 9 T r ansformers, elements in disguise rese arch teams t argeted t he gene encoding hypo xanthine phos- phoribosyl transferase ( Hpr t ) by homologo us recom binatio n in embry- onic stem (ES) cells. These cells were chosen for their uniq ue potential to be manip ulated in vitro and rein troduced into mo use blastoc ysts, producin g chimeric animals that ca n transmit the new traits to su bsequent genera tions. Kirk Thomas and M ario C apecchi engineered tw o classes of vectors tha t efficiently disrupted Hpr t either by replacing endog enous sequences with the exogenous neo my cin-resistance gene or b y inser ting the exogenous sequence into the Hpr t locus. The y then identified ES cells carrying mu tated genes b y selecting for acquired r esist ance to the drug G418 and the base analogue 6-TG. Oli ver Smithies and co-w orker s also used this technique to co r rect the defects of three independent Hpr t -muta ted ES cell lines. T ogether , these ground- breaking st udies paved the wa y for functional genomics a nd gene therap y . Fr ancesc a Cesari, Locum Associ ate Editor , Natu re ORIGINAL RESEARCH P APERS Rubin, G. M. & Spradling, A. C. Genetic transformation of Drosophila with transposable element vectors. Science 218 , 348–353 (1982) | Spr adling, A. C. & Rubin, G. M. T ransposition of cloned P elements into Dr osophila germ line chromosomes. Science 218 , 341–347 (1982) | Doetschman, T . et al. T argetted correction of a mutant HPR T gene in mouse embryonic stem cells. Nature 330 , 576–578 (1987) | Thomas, K. R. & Capecchi, M. R. Site-directed mutagenesis by gene targeting in mouse embryo-derived stem cells. Cell 51 , 503–512 (1987) FUR THER READING Hinnen, A. et al. T ransformation of yeast. Proc. Natl Acad. Sci. USA 75 , 1929–1933 (1978) | Capecchi, M. R. High efficiency transformation by direct microinjection of DNA into cultured mammalian cells. Cell 22 , 479–488 (1980) S10 | OCTOBER 2007 ww w.natur e.com/ milestones/ dnatechnologies © 2007 N a ture Publi s hi n g G r o u p Whereas r andomness is avoided in most experimental techniques, it is fundamental to sequencing approaches. In the r ace to sequence the human genome, resear ch groups had to choose between the random whole-genome shotgun sequencing approach or the mor e order ed map-based sequencing approach. When Frederick Sanger and colleagues sequenced the 48-kb bacteriophage λ genome in 1982, the community was still undecided as to whether directed or r andom sequencing strategies wer e better . With directed strategies, DNA sequences were br oken down into order ed and overlapping fragments to build a map of the genome, and these fragments wer e then cloned and sequenced. With the shotgun approach, DNA sequences wer e broken randomly, cloned, sequenced and then pieced together by analysing the overlap. Sanger et al. compared these str ategies while sequencing bacteriophage λ and reported that the random approach was faster than any dir ected method. One problem with the r andom approach, however , was that of filling gaps when the sequence was nearly complete (or closure), as randomly selected clones wer e often redundant. For instance, Sanger et al. used — in their opinion mistakenly — direct sequencing str ategies to finish the last 10% of the bacteriophage λ sequence. In 1991, Al Edwards and Thomas Caskey proposed a method to maximize efficiency by minimizing gap formation and redundancy: sequence both ends (but not the middle) of a long clone, rather than the entir ety of a short clone. Although the shotgun approach was now accepted for sequencing short stretches of DNA, map-based techniques were still consider ed necessary for large genomes. Like the dir ected strategies, map-based sequencing subdivided the genome into order ed 40-kb fragments, which were then sequenced using the shotgun approach. In 1995, however , Robert Fleischmann and colleagues used a whole-genome shotgun approach to sequence the 1,800-kb genome of Haemophilus influenzae — the first complete genome of a free-living or ganism. The authors had randomly gener ated large 40-kb fragments and had thereby bypassed the mapping stage. In doing so, they had proved that genome-assembly progr ammes that matched overlap were reliable and that whole-genome shotgun sequencing worked, in principle. The H. influenzae genome, however , was a mere DNA fragment compar ed with the 1,500-fold longer ~3 billion base-pair human genome. In 1996, Craig V enter and colleagues proposed that the whole-genome shotgun approach could be used to sequence the human genome owing to two factors: its past successes in assembling genomes and the development of bacterial artificial chromosomes (BAC) libr aries, which allowed large fr agments of DNA to be cloned. A showdown ensued, with the biotechnology firm Celera Genomics wielding whole-genome shotgun sequencing and the International Human Genome Sequencing Consortium wielding map-based sequencing. Y et, when the dust settled, it was a draw — both gr oups published their initial drafts of the human genome concurrently in 2001. Asher Mullard, Assistant Editor , Nature Reviews Micr obiology and Nature Reviews Molecular Cell Biology ORIGINAL RESEARCH P APERS Sanger , F . et al. Nucleotide sequence of bacteriophage λ DNA. J. Mol. Biol. 162 , 729–773 (1982) | Edwards, A. & Caskey, C. T . Closure strategies for random DNA sequencing. Methods 3 , 41–47 (1991) | Fleischmann, R. D. et al. Whole-genome random sequencing and assembly of Haemophilus influenzae Rd. Science 269 , 496–512 (1995) | V enter , J. C. et al. A new strategy for genome sequencing. Natur e 381 , 364–366 (1996) MILEST ONE 10 Randomness versus or der T oday , it would be difficult to conceive o f a biolog y labora tory withou t a polymerase chain reac- tion a ppara tus—‘the PCR machine ’ . Standa rd molecular p rac tices that we ta ke for g ranted, su ch as c reating cons tructs for expr essing tagged pro teins, amplifying genes fro m minu te samp les for clo ning and intr oducing m uta tions in to genes, would be a com pletely differen t story , a much mo re complica ted one, wi t hou t the adven t of PCR. This technique, which has revo lutio nized biological la b ora tory researc h, was first developed about two decades ago . Y et in 1971, in a Jo urnal of M olecular Bi ology rep or t, Ha r Gobind Khoran a and colleagues had already described a process called repair r eplication fo r synthe- sizing short DN A duplexes a nd single-stranded DN A by polymer - ases. This report outlined several feat ures that a re hallmarks of PCR, but f ell short of an experimen tal test. I t predicted, fo r examp le, that MILEST ONE 11 Chain r eaction Daniel Jones In the race t o sequence the human genome , r esearch groups had t o choose between the random whole-genome shotgun sequencing approach or the more or dered map-based sequencing approach. ▼ NA TURE MILEST ONES | DNA TECHNOL OGIES O CTOBER 2007 | S11 © 2007 N a ture Publi s hi n g G r o u p the DN A duplex wo uld have t o be denat ured to single strands, tha t an excess of p r imer to tem pla te would be requir ed to ov ercome seconda r y structures genera ted by single- stranded tem plate and tha t, following com pletion o f the reaction b y DN A polymerase, the cycle would ha ve to be reinitia ted if the templa te duplex had renatu red. However , it to ok anot her a lmost 17 years befo re PCR, as we know i t, was described. Kary Mullis an d Fr ed Faloona, in a 1987 pa per in Me t h o d s in Enzymology , experimentally defined the basic steps of PCR. The au thors specula ted about th e poten tial ap plicatio ns of the method, man y of which ar e now r outine molecular biology proced ures. I nter estingly , 2 years b efore thi s land mark rep or t was published, N orman Arnheim and colleagues demonstra ted the p ower of PCR as a diagnostic tool by sho w- ing that suc h an ap proac h could be used to rap idly am plif y the β -globin sequence from clinical sam ples to determine whether it possess ed the mu tation for sickle cell an aemia. I t was not until 1988 that H enr y Erlich and colleagues described the use of a thermostable DN A poly meras e f rom Thermus aqua ticus , which was a key innova tion that allowed a nne aling and ext ension at high tem p eratur es. This cr ucial imp ro vement did a way with the need to replenish the enzyme a fter each cycle, increased specificity , yield and sensitivi ty , allow ed the process to be used to generat e longer p roducts and, finally , paved the way f or its a utoma- tion. PCR, as we know i t, was born. Sowmya S wami nat han, Sen ior Edi tor , Na ture Cell Biology ORIGINAL RESEARCH P APERS Kleppe, K., Ohtsuka, E., Kleppe, R., Molineux, I. & Khorana, H. G. Studies on polynucleotides. XCVI. Repair replications of short synthetic DNAs as catalyzed by DNA polymerases. J. Mol. Biol. 56 , 341–361 (1971) | Saiki, R. K. et al. Enzymatic amplification of β -globin genomic sequences and r estriction site analysis for diagnosis of sickle cell anemia. Science 230 , 1350–1354 (1985) | Mullis, K. B. & Faloona F . A. Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. Meth. Enzymol. 155 , 335–350 (1987) | Saiki, R. K. et al. Primer -directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239 , 487–491 (1988) ▼ Following the discovery of the DNA structure in the 1950s, attention gradually shifted to the next big thing: finding those parts of the human genome that differed between individuals. In 1985, Alec Jeffreys, Victoria Wilson and Swee Lay Thein described a highly variable segment of DNA that would help in this quest: the minisatellite. This was not only a sensitive tool for human and other genetic studies but it also had applications in person identification, which opened it up for use in paternity analysis, immigration disputes and for ensic science. Molecular markers were alr eady being used in linkage studies, for example, and in antenatal diagnosis. Y et the variability of these at-best dimorphic markers — made of DNA segments with variable length that were cr eated by endonucleases — would not stretch to give the resolution needed to distinguish individuals easily . In 1984, a tandemly arranged 33 base-pair sequence had been detected in an intron of the human myoglobin gene; the basis of the 1985 paper was the realization that a probe derived fr om this kind of repeat could pick up many variable-length segments in the genome simply by Southern blotting. Because of their repeated natur e such sequences, or minisatellites, are pr one to expansion and contraction, and so each locus can vary among individuals. Each minisatellite has a ‘core’ motif of 6–100 base pairs: if more than one cor e sequence was used then a ‘profile’ or ‘fingerprint’ of an individual could be obtained that was, in effect, unique — as the paper reported, this allowed even close relatives to be unambiguously identified. The authors were awar e of the wide potential applications of ‘DNA fingerprinting’, although it is unlikely that they had predicted its runaway success in fields ranging fr om conservation biology to forensics. Minisatellites made their first appearance in court that same year , in an immigration dispute over the identity of a young boy returning to the UK fr om Ghana. Minisatellite profiling of the boy’ s alleged mother and three siblings confirmed the relationship claim made by the defence, and he was granted permission to r emain in the country . T oday, DNA fingerprinting is alive and well; if anything, it has grown in recognition. However , the name is one of the few things that have been preserved, given the lar ge changes to the field brought about by new markers and technologies — not least the invention of PCR. T anita Casci, Senior Editor , Nature Reviews Genetics ORIGINAL RESEARCH P APERS Jeffreys, A. J., Wilson, V . & Thein, S. L. Hypervariable ‘minisatellite’ regions in human DNA. Nature 314 , 67–73 (1985) | Jeffr eys, A. J., Brookfield, J. F . & Semeonoff, R. P ositive identification of an immigration test-case using human DNA fingerprints. Natur e 317 , 818–819 (1985) T oday , it would be difficult to conceive of a biology laborat ory without a polymerase chain reaction apparatus— ‘the PCR machine’ . MILEST ONE 12 A r epeat success MILEST ONES S12 | OCTOBER 2007 ww w.natur e.com/ milestones/ dnatechnologies © 2007 N a ture Publi s hi n g G r o u p G The year 1987 marked a quantum leap for DNA cloning with a tenfold increase in vector capacity . A vector carrying a 50-kb insert, the largest available at the time, was useful for examining a single gene but far too small to also contain all regulatory r egions. There was also gr owing interest in constructing compr ehensive libraries covering the whole genomes of higher organisms — a supr emely daunting task if undertaken in 50 kb fragments. T o increase capacity, Maynar d Olson and colleagues at W ashington University exchanged the classical cloning vehicle, a circular Escherichia coli plasmid, for what they called a yeast artificial chromosome (Y AC): a linear DNA molecule that mimics a yeast chromosome, complete with centromer e and telomeres. All necessary Y AC elements were incorporated into cir cular plasmids that could be linearized in vitro during insertion of the exogenous DNA. The resulting linear fr agment, carrying as much as several hundr ed kb of foreign DNA, faithfully replicated in yeast. Y ACs were eagerly adopted by the scientific community, particularly to establish physical maps of whole genomes. Y et, with their increased use, some problems surfaced: many clones turned out to be chimaer as of noncontiguous DNA fragments, inserts wer e occasionally unstable and purification of Y ACs proved challenging due to contamination from endogenous yeast chromosomes. P eople started to miss the simplicity of a bacterial cloning system. In 1992, the time was ripe to revisit E. coli with the aim of adapting it for large-fr agment cloning. A group at the California Institute of T echnology led by Melvin Simon modified an endogenous circular plasmid in E. coli , the fertility (F) factor present at one or two copies per cell, to create a cloning vector . In refer ence to its yeast cousin, they called it bacterial artificial chromosome (BAC). With a cloning capacity of ~300 kb, BACs are not as potent as Y ACs, but they have all the advantages of a bacterial vector: stability, and ease of manipulation and purification. T oday Y ACs are still in use; however , BACs have become the workhorses in genomic resear ch for any application that requir es large DNA inserts. Nicole Rusk, Associate Editor , Nature Methods MILEST ONE 13 Size matters MILEST ONE 14 ChIPping away at pr otein–DNA inter actions W e now kno w that chr oma tin is not simp ly an inert packaging structure for D NA, bu t pro vides a dynamic enviro nment with an im portant ro le in regulatin g processes such as gene tra n- scription, D N A repair and r eplicatio n. Ho wever , over m uch of the twentieth cen tur y the study of chr oma tin progr essed more slowly than tha t of DN A, often because a ppr opria te tools wer e not available. The chro matin imm unopr e cipi tation (ChIP) assa y has become an indispensable tool, b ut the req uired techniques took man y years to evolve. The assa y allows transcription factors, chr omatin p rot eins (such as histones) o r post- transla t ional modifications t o these prot eins to be map ped to specific regions o f genomic DN A. A crucial aspec t of the ChIP assay is to p reserve physio logically relevan t interactions between DN A and chroma tin protein s, which was made possible by crosslinkin g methods. Fo rmaldehyde cr osslinking be came popular as i t works well with histo nes and is easily reversib le. In an earl y report in 1978, J ackson used formaldeh yde crosslinking and electro- phor esis to demonstra te histone–DN A and histone–histon e interaction s in isolated n uclei. Some years lat er , V arsha vsky , Lis and their colleagues p ublished influen tial papers in which an im munopre cipitat ion s tep was intro duc ed w ith sp ec if ic histone a ntibodies, which was performed after pr otein–DN A com plexes had been formaldeh yde or ultra violet crosslinked and shear ed. This app roach allowed in vestigator s to show , for exam ple, that chro matin a t the heat-shock pro tein 70 promoter of Droso ph ila was perturbed upon heat shock. Neil Smith ▼ ORIGINAL RESEARCH P APERS Burke, D. T . et al. Cloning of large segments of exogenous DNA into yeast by means of artificial chromosome vectors. Science 236 , 806–811 (1987) | Shizuya, H. et al. Cloning and stable maintenance of 300- kilobase-pair fragment of human DNA in Escherichia coli using an F-factor -based vector . Proc. Natl Acad. Sci. USA 89 , 8794–8797 (1992) NA TURE MILEST ONES | DNA TECHNOL OGIES O CTOBER 2007 | S13 © 2007 N a ture Publi s hi n g G r o u p The generatio n of genome sequences from a wide ran ge of o rganisms has opened the field of com parative genomic anal ysis, assisting the anno- tation o f individual genomes, and bringing new insigh ts into g enome evolu tion. Central to the field of com parative g enomics hav e been progra ms used to s earch a nd align pro tein or DN A s equences based on a measure of simila rity . Sequence alignment can in volve either global alignmen t, in which the two sequences are aligned o ver their entir e length, or local align- ment, in which sub region s of the two sequences are aligned . The lat ter has been more widely a pplied, as DN A sequences generally show isola ted regio ns of simila rity . An exact solution t o the global alignment p roblem was develo ped by Sa ul Needleman a nd Christian W unsch in 1970, by a pplyin g dynamic progra mming to find the optimal alignment between tw o sequences. In 1981, T emple Smith an d Michael W aterma n extended this dynamic progra mming app roach to solv e the local alignment pr oblem. These exact solutio ns placed sequence com pari- son s on a firm m ath ema tical gro und- ing, and f ormed the basis for the earl y alignment sear ch algo rithms. Ho wever , the exact solutio ns pro ved slow in practice, especially fo r searching la rge da tabases, spurring the developmen t of faster heuri stic ap proach es. One early successful heu- ristic algorithm to enable efficient searchin g of larg e databases, F AST A, was presen ted by David Lipma n and W illiam Pea rson in 1988. This simplified the pr oblem by searching MILEST ONE 15 BLAST -off for genomes ▼ A further key advance was the develop ment o f s elective an tib odies, such as those to modified histones, for im munopre cipit ati ng sp e cif ic pro tein–DNA co mplexes. In the la te 1980s, ChIP with these an tibodies pro vided a func tional link between histone acetyla tion and tran scription. Later , PCR was us ed to am plify the DN A purified from the an tib ody com plexes; ho wever , with the adven t of DN A microarra ys, the ChIP assay was drama tically extended to loca te transcriptio n factor bindin g sites on a genome-wide scale, a techniq ue that acquired the mo niker ‘ ChIP-c hip ’ . In two p ioneering papers, Ren, I yer and their colleagues co mbined ChIP of tran scription factor s in yeast with a PCR step to am plif y and label the immu noprec ipitate d DNA b efore hybridiza tion to microarrays o f gene pro moters. A genome-wide view of the sites bound b y transcriptio n fac- tors under va rious cellula r condition s could now be co mbined wi th micro- array g ene expressio n analysis to determine the direct function of these factors. Since then, ChIP -chip has also allow ed mapp ing of c hro ma tin pro teins and histo ne mo difications across the genome , and so has pro ved a powerful tool to in vestigat e the influence of chro matin on gene tran- scription an d other DN A processes. Alex Ec clest on, Senio r Edit or , Natu re ORIGINAL RESEARCH P APERS Solomon, M. J., Larsen, P . L. & Varshavsky, A. Mapping pr otein– DNA interactions in vivo with formaldehyde: evidence that histone H4 is retained on a highly transcribed gene. Cell 53 , 937–947 (1988) | Ren, B. et al. Genome-wide location and function of DNA binding proteins. Science 290 , 2306–2309 (2000) | Iyer , V . R. et al. Genomic binding sites of the yeast cell-cycle transcription factors SBF and MBF . Nature 409 , 533–538 (2001) FUR THER READING Jackson, V . Studies on histone organization in the nucleosome using formaldehyde as a reversible cross-linking agent. Cell 15 , 945–954 (1978) | Gilmour , D. S. & Lis, J. T . Detecting protein–DNA interactions in vivo : distribution of RNA polymerase on specific bacterial genes. Proc. Natl Acad. Sci. USA 81 , 4275–4279 (1984) | Hebbes, T . R., Thorne, A. W . & Crane-Robinson, C. A direct link between cor e histone acetylation and transcriptionally active chromatin. EMBO J . 7 , 1395–1402 (1988) | Braunstein, M., Rose, A. B., Holmes, S. G., Allis, C. D. & Broach, J. R. T ranscriptional silencing in yeast is associated with reduced nucleosome acetylation. Genes Dev . 7 , 592–604 (1993) | Strahl-Bolsinger , S., Hecht, A., Luo, K. & Grunstein, M. SIR2 and SIR4 interactions differ in core and extended telomeric heterochromatin in yeast. Genes Dev . 11 , 83–93 (1997) | Kuo, M. H. & Allis, C. D. In vivo cross-linking and immunoprecipitation for studying dynamic protein:DNA associations in a chromatin environment. Methods 19 , 425–433 (1999) …BLAST inde xes the query sequence and scans against a database . MILEST ONES S14 | OCTOBER 2007 ww w.natur e.com/ milestones/ dnatechnologies © 2007 N a ture Publi s hi n g G r o u p

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