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Foundations of Molecular Cloning – Past, Present & FutureRebecca Tirabassi, Bitesize Bio.IntroductionMolecular cloning refers to the isolation of aDNA sequence from any species (often a gene),and its insertion into a vector for propagation,without alteration of the original DNA sequence.Once isolated, molecular clones can be used togenerate many copies of the DNA for analysisof the gene sequence, and/or to express theresulting protein for the study or utilization ofthe protein’s function. The clones can also bemanipulated and mutated in vitro to alter theexpression and function of the protein.The basic cloning workflow includes four steps:1. Isolation of target DNA fragments(often referred to as inserts)2. Ligation of inserts into an appropriate cloningvector, creating recombinant molecules(e.g., plasmids)3. Transformation of recombinant plasmids intobacteria or other suitable host for propagation4. Screening/selection of hosts containing theintended recombinant plasmidThese four ground-breaking steps were carefullypieced together and performed by multiplelaboratories, beginning in the late 1960s andearly 1970s. A summary of the discoveriesthat comprise traditional molecular cloning isdescribed in the following pages.The Foundation of Molecular CloningCutting (Digestion).Recombinant DNA technology first emerged inthe late 1960s, with the discovery of enzymesthat could specifically cut and join doublestranded DNA molecules. In fact, as early as1952, two groups independently observedthat bacteria encoded a “restriction factor” thatprevented bacteriophages from growing withincertain hosts (1,2). But the nature of the factorwas not discovered until 1968, when Arber andLinn succeeded in isolating an enzyme, termed arestriction factor, that selectively cut exogenousDNA, but not bacterial DNA (3). These studiesalso identified a methylase enzyme that protectedthe bacterial DNA from restriction enzymes.Shortly after Arber and Linn’s discovery, Smithextended and confirmed these studies byisolating a restriction enzyme from Haemophilusinfluenza. He demonstrated that the enzymeFigure 1. Traditional Cloning WorkflowDNA PreparationMCSFragment A(PCR-amplified orannealed oligos) Vector BMCSORVector A nt AVector B LigationDigestedVector BDigestedVector A DigestedVector BDNA ligaseTransformationAssembledDNAUsing PCR, restriction sites are added to both ends of a dsDNA, which is then digested by the corresponding restriction enzymes(REases). The cleaved DNA can then be ligated to a plasmid vector possessing compatible ends. DNA fragments can also bemoved from one vector into another by digesting with REases and ligating with compatible ends of the target vector. Assembledconstruct can then be transformed into Escherichia coli (E. coli).selectively cut DNA in the middle of a specific6 base-pair stretch of DNA; one characteristic ofcertain restriction enzymes is their propensity tocut the DNA substrate in or near specific, oftenpalindromic, “recognition” sequences (4).The full power of restriction enzymes wasnot realized until restriction enzymes and gelelectrophoresis were used to map the SimianVirus 40 (SV40) genome (5). For these seminalfindings, Werner Arber, Hamilton Smith, andDaniel Nathans shared the 1978 Nobel Prize inMedicine.FEATURE ARTICLEMolecular cloning, a term that has come to mean the creation of recombinant DNA molecules, has spurred progress throughout thelife sciences. Beginning in the 1970s, with the discovery of restriction endonucleases – enzymes that selectively and specifically cutmolecules of DNA – recombinant DNA technology has seen exponential growth in both application and sophistication, yieldingincreasingly powerful tools for DNA manipulation. Cloning genes is now so simple and efficient that it has become a standardlaboratory technique. This has led to an explosion in the understanding of gene function in recent decades. Emerging technologiespromise even greater possibilities, such as enabling researchers to seamlessly stitch together multiple DNA fragments and transformthe resulting plasmids into bacteria, in under two hours, or the use of swappable gene cassettes, which can be easily moved betweendifferent constructs, to maximize speed and flexibility. In the near future, molecular cloning will likely see the emergence of a newparadigm, with synthetic biology techniques that will enable in vitro chemical synthesis of any in silico-specified DNA construct.These advances should enable faster construction and iteration of DNA clones, accelerating the development of gene therapy vectors,recombinant protein production processes and new vaccines.Assembling (Ligation).Much like the discovery of enzymes that cutDNA, the discovery of an enzyme that could joinDNA was preceded by earlier, salient observations.In the early 1960s, two groups discovered thatgenetic recombination could occur though thebreakage and ligation of DNA molecules (6,7),closely followed by the observation that linearbacteriophage DNA is rapidly converted tocovalently closed circles after infection of the host(8). Just two years later, five groups independentlyisolated DNA ligases and demonstrated theirability to assemble two pieces of DNA (9-13).Not long after the discovery of restrictionenzymes and DNA ligases, the first recombinantDNA molecule was made. In 1972, Bergseparately cut and ligated a piece of lambdabacteriophage DNA or the E. coli galactose operonwith SV40 DNA to create the first recombinantDNA molecules (14). These studies pioneeredthe concept that, because of the universalnature of DNA, DNA from any species could bejoined together. In 1980, Paul Berg shared theNobel Prize in Chemistry with Walter Gilbertand Frederick Sanger (the developers of DNAsequencing), for “his fundamental studies of thebiochemistry of nucleic acids, with particularregard to recombinant DNA.”Transformation.Recombinant DNA technology would be severelylimited, and molecular cloning impossible,without the means to propagate and isolate thenewly constructed DNA molecule. The abilityto transform bacteria, or induce the uptake,incorporation and expression of foreign geneticmaterial, was first demonstrated by Griffith whenhe transformed a non-lethal strain of bacteria intoa lethal strain by mixing the non-lethal strain with03

feature article continued for cloning were cumbersome, difficult to workwith and limited in number, and experimentswere limited by the amount of insert DNA thatcould be isolated. Research over the next fewdecades led to improvements in the techniquesand tools available for molecular cloning.FEATURE ARTICLEheat-inactivated lethal bacteria (15). However,the nature of the “transforming principle” thatconveyed lethality was not understood until1944. In the same year, Avery, Macleod andMcCarty demonstrated that DNA, and notprotein, was responsible for inducing the lethalphenotype (16).allowed researchers to screen for bacterialcolonies containing plasmids with the foreignDNA insert. When bacteria were plated onthe correct media, white colonies containedplasmids with inserts, while blue coloniescontained plasmids with no inserts. pUCplasmids had an additional advantage overexisting vectors; they contained a mutationthat resulted in higher copy numbers, thereforeincreasing plasmid yields.Early vector design.Development of the first standardized vector.Scientists working in Boyer’s lab recognizedthe need for a general cloning plasmid, acompact plasmid with unique restrictionsites for cloning in foreign DNA and theexpression of antibiotic resistance genes forselection of transformed bacteria. In 1977, theydescribed the first vector designed for cloningpurposes, pBR322 (20). This vector was small, 4 kilobases in size, and had two antibioticresistance genes for selection.Initially, it was believed that the commonbacterial laboratory strain, E. coli, wasrefractory to transformation, until Mandel andHiga demonstrated that treatment of E. coliwith calcium chloride induced the uptake ofbacteriophage DNA (17). Cohen applied thisprinciple, in 1972, when he pioneered thetransformation of bacteria with plasmids toconfer antibiotic resistance on the bacteria (18).The ultimate experiment: digestion, ligationand transformation of a recombinant DNAmolecule was executed by Boyer, Cohenand Chang in 1973, when they digestedthe plasmid pSC101 with EcoRI, ligatedthe linearized fragment to another enzymerestricted plasmid and transformed the resultingrecombinant molecule into E. coli, conferringtetracycline resistance on the bacteria (19), thuslaying the foundation for most recombinantDNA work since.Building on the GroundworkWhile scientists had discovered and appliedall of the basic principles for creating andpropagating recombinant DNA in bacteria,the process was inefficient. Restriction enzymepreparations were unreliable due to nonstandardized purification procedures, plasmidsImproving restriction digests.Vectors with on-board screening and higher yields.Although antibiotic selection prevented nontransformed bacteria from growing, plasmidsthat re-ligated without insert DNA fragments(self-ligation) could still confer antibioticresistance on bacteria. Therefore, finding thecorrect bacterial clones containing the desiredrecombinant DNA molecule could be timeconsuming.Early work with restriction enzymes washampered by the purity of the enzymepreparation and a lack of understanding ofthe buffer requirements for each enzyme. In1975, New England Biolabs (NEB) becamethe first company to commercialize restrictionenzymes produced from a recombinant source.This enabled higher yields, improved purity,lot-to-lot consistency and lower pricing.Currently, over 4,000 restriction enzymes,recognizing over 300 different sequences, havebeen discovered by scientists across the globe[for a complete list of restriction enzymes andrecognition sequences, visit REBASE at rebase.neb.com (22)]. NEB currently supplies over230 of these specificities.Vieira and Messing devised a screening toolto identify bacterial colonies containingplasmids with DNA inserts. Based upon thepBR322 plasmid, they created the series ofpUC plasmids, which contained a “blue/white screening” system (21). Placement of amultiple cloning site (MCS) containing severalunique restriction sites within the LacZ geneNEB was also one of the first companies todevelop a standardized four-buffer system, andto characterize all of its enzyme activities in thisbuffer system. This led to a better understanding of how to conduct a double digest, or thedigestion of DNA with two enzymes sim-ultaneously. Later research led to the developmentof one-buffer systems, which are compatibleA History of Molecular Cloning1971Restriction enzymemapping of thesimian virus SV40 (5)1961Genetic recombinationdemonstrated (6,7)1977Report of the firstcloning vector(pBR322) (20)1974NEB opensfor business1970Isolation ofrestrictionenzymes thatselectively cut (4)1978Nobel Prizeawarded to Smith,Arber and Nathansfor the discovery ofrestriction enzymes51971952 – 53Genetic demonstrationof phage restriction(1,2)1975Launch of REBASE(Restriction tion of thefirst DNA ligases(9–13)1968Isolation of the firstrestriction factor thatcould selectively cutbacteriophage DNA (3)1976 – 77Introduction ofMaxam-Gilbert andSanger sequencing(26,27)1972Assembly of the firstrecombinant DNA&Tranformation of thefirst E. coli (17,18)

with the most common restriction enzymes(such as NEB’s CutSmart Buffer).With the advent of commercially availablerestriction enzyme libraries with knownsequence specificities, restriction enzymesbecame a powerful tool for screening potentialrecombinant DNA clones. The “diagnosticdigest” was, and still is, one of the most common techniques used in molecular cloning.Vector and insert preparation.The CIP enzyme proved difficult to inactivate,and any residual activity led to dephosphorylation of insert DNA and inhibition of the ligation reaction. The discovery of the heat-labilealkaline phosphatases, such as recombinantShrimp Alkaline Phosphatase (rSAP) andAntarctic Phosphatase (AP) (both sold by NEB),decreased the steps and time involved, as a simple shift in temperature inactivates the enzymeprior to the ligation step (25).DNA sequencing arrives.DNA sequencing was developed in the late1970s when two competing methods wereThe ability to determine the sequence of astretch of DNA enhanced the reliability andversatility of molecular cloning. Once cloned,scientists could sequence clones to definitivelyidentify the correct recombinant molecule,identify new genes or mutations in genes, andeasily design oligonucleotides based on theknown sequence for additional experiments.The impact of the polymerase chain reaction.One of the problems in molecular cloning inthe early years was obtaining enough insertDNA to clone into the vector. In 1983,Mullis devised a technique that solved thisproblem and revolutionized molecular cloning(28). He amplified a stretch of target DNAby using opposing primers to amplify bothcomplementary strands of DNA, simultaneously.Through cycles of denaturation, annealingand polymerization, he showed he couldexponentially amplify a single copy of DNA.The polymerase chain reaction, or PCR, madeit possible to amplify and clone genes frompreviously inadequate quantities of DNA. Forthis discovery, Kary Mullis shared the 1993Nobel Prize in Chemistry “for contributions1991Introduction of USER cloning (36)to the developments of methods within DNAbased chemistry”.In 1970, Temin and Baltimore independentlydiscovered reverse transcriptase in viruses, anenzyme that converts RNA into DNA (29,30).Shortly after PCR was developed, reversetranscription was coupled with PCR (RT-PCR)to allow cloning of messenger RNA (mRNA).Reverse transcription was used to create a DNAcopy (cDNA) of mRNA that was subsequentlyamplified by PCR to create an insert forligation. For their discovery of the enzyme,Howard Temin and David Baltimore wereawarded the 1975 Nobel Prize in Medicineand Physiology, which they shared withRenato Dulbecco.Cloning of PCR products.The advent of PCR meant that researcherscould now clone genes and DNA segmentswith limited knowledge of amplicon sequence.However, there was little consensus as to theoptimal method of PCR product preparationfor efficient ligation into cloning vectors.Several different methods were initially usedfor cloning PCR products. The simplest, andstill the most common, method for cloningPCR products is through the introductionof restriction sites onto the ends of the PCRproduct (31). This allows for direct, directionalcloning of the insert into the vector afterrestriction digestion. Blunt-ended cloning wasdeveloped to directly ligate PCR productsgenerated by polymerases that produced bluntends, or inserts engineered to have restrictionFEATURE ARTICLECloning efficiency and versatility were alsoimproved by the development of different techniques for preparing vectors prior to ligation.Alkaline phosphatases were isolated that couldremove the 3 and 5 phosphate groups fromthe ends of DNA [and RNA; (23)]. It was soondiscovered that treatment of vectors with CalfIntestinal Phosphatase (CIP) dephosphorylatedDNA ends and prevented self-ligation of thevector, increasing recovery of plasmids withinsert (24).devised. Maxam and Gilbert developed the“chemical sequencing method,” which relied onchemical modification of DNA and subsequentcleavage at specific bases (26). At the sametime, Sanger and colleagues published on the“chain-termination method”, which becamethe method used by most researchers (27). TheSanger method quickly became automated,and the first automatic sequencers were sold in1987.2007Introduction of ligationindependentcloning (35)201990001985201019951987First automatedsequencersbecome available1983Introductionof PCR (28)20051993Mullis shares theNobel Prize for hiswork in PCR1985Generation ofpUC plasmids(21)2000in vitro synthesisof a whole genome(41,42)2008Synthesis of themycoplasma genitaliumgenome using GibsonAssembly (39)&Introduction ofHigh-Fidelity (HF )restriction enzymes2010First synthetic life –Mycoplasma mycoides(45)05

feature article continued FEATURE ARTICLEsites that left blunt ends once the insert wasdigested. This was useful in cloning DNAfragments that did not contain restriction sitescompatible with the vector (32).Shortly after the introduction of PCR, overlapextension PCR was introduced as a method toassemble PCR products into one contiguousDNA sequence (33). In this method, the DNAinsert is amplified by PCR using primersthat generate a PCR product containingoverlapping regions with the vector. Thevector and insert are then mixed, denaturedand annealed, allowing hybridization of theinsert to the vector. A second round of PCRgenerates recombinant DNA molecules ofinsert-containing vector. Overlap extensionPCR enabled researchers to piece togetherlarge genes that could not easily be amplifiedby traditional PCR methods. Overlap extensionPCR was also used to introduce mutations intogene sequences (34).Development of specialized cloningtechniques.In an effort to further improve the efficiency ofmolecular cloning, several specialized tools andtechniques were developed that exploited theproperties of unique enzymes.TA Cloning.One approach took advantage of a propertyof Taq DNA Polymerase, the first heat-stablepolymerase used for PCR. During amplification,Taq adds a single 3 dA nucleotide to the endof each PCR product. The PCR product canbe easily ligated into a vector that has been cutand engineered to contain single T residues oneach strand. Several companies have marketedthe technique and sell kits containing cloningvectors that are already linearized and “tailed”.LIC.Ligation independent cloning (LIC), as itsname implies, allows for the joining of DNAmolecules in the absence of DNA ligase.LIC is commonly performed with T4 DNAPolymerase, which is used to generate singlestranded DNA overhangs, 12 nucleotideslong, onto both the linearized vector DNAand the insert to be cloned (35). When mixedtogether, the vector and insert anneal throughthe long stretch of compatible ends. The lengthof the compatible ends is sufficient to holdthe molecule together in the absence of ligase,even during transformation. Once transformed,the gaps are repaired in vivo. There are severaldifferent commercially available products forLIC.USER cloning.USER cloning was first developed in the early1990s as a restriction enzyme- and ligaseindependent cloning method (36). When firstconceived, the method relied on using PCRprimers that contained a 12 nucleotide 5 tail,in which at least four deoxythymidine baseshad been substituted with deoxyuridines. ThePCR product was treated with uracil DNAglycosidase (UDG) and Endonuclease VIII,Figure 2. Overview of PCRReaction Assembly3 DNA region5 of interest5 3 Amplification reactioncomponents are combinedand added to the thermocycler98 C5 DenaturationTemperature is increasedto separate DNA strands3 3 5 5 Temperature is decreasedto allow primers to base pairto complementary DNA template5 3 Primer3 3 Primer3 5 5 TemplateDNA strands68 to 72 CExtensionPolymerase extends primerto form nascent DNA strandExponentialAmplificationProcess is repeated,and the region of interestis amplified exponentially1st cycle5 3 3 5 5 3 3 5 2nd cycle3rd cycle4th cycle22 4 copies23 8 copies24 16 copies0625 32 copiesFuture TrendsMolecular cloning has progressed from thecloning of a single DNA fragment to theassembly of multiple DNA components intoa single contiguous stretch of DNA. Newand emerging technologies seek to transformcloning into a process that is as simple asarranging “blocks” of DNA next to each other.DNA assembly methods.Many new, elegant technologies allow forthe assembly of multiple DNA fragments ina one-tube reaction. The advantages of thesetechnologies are that they are standardized,seamless and mostly sequence independent.In addition, the ability to assemble multipleDNA fragments in one tube turns a series ofpreviously independent restriction/ligationreactions into a streamlined, efficient procedure.Different techniques and products for geneassembly include SLIC (Sequence and LigaseIndependent Cloning), Gibson Assembly (NEB),GeneArt Seamless Cloning (Life Technologies)and Gateway Cloning (Invitrogen) (35,37,38).In DNA assembly, blocks of DNA to beassembled are PCR amplified. Then, the DNAfragments to be assembled adjacent to oneanother are engineered to contain blocks ofcomplementary sequences that will be ligatedtogether. These could be compatible cohesiveends, such as those used for Gibson Assembly,or regions containing recognition sites forsite-specific recombinases (Gateway). Theenzyme used for DNA ligation will recognizeand assemble each set of compatible regions,creating a single, contiguous DNA molecule inone reaction.Synthetic biology.48 to 72 CAnnealingwhich excises the uracil bases and leaves a3 overlap that can be annealed to a similarlytreated vector. NEB sells the USER enzymefor ligase and restriction enzyme independentcloning reactions.NascentDNA strands30th cycle231 2 billion copiesDNA synthesis is an area of synthetic biologythat is currently revolutionizing recombinantDNA technology. Although a complete genewas first synthesized in vitro in 1972 (40),DNA synthesis of large DNA molecules did notbecome a reality until the early 2000s, whenresearchers began synthesizing whole genomesin vitro (41,42). These early experimentstook years to complete, but technology isaccelerating the ability to synthesize large DNAmolecules.ConclusionIn the last 40 years, molecular cloning hasprogressed from arduously isolating andpiecing together two pieces of DNA, followedby intensive screening of potential clones, toseamlessly assembling up to 10 DNA fragmentswith remarkable efficiency in just a few hours,or designing DNA molecules in silico and

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