Molecular cloning, a term that has come to mean the creation of recombinant DNA molecules, has spurred progress throughout the life sciences. Beginning in the 1970s, with the discovery of restriction endonucleases – enzymes that selectively and specifically cut molecules of DNA – recombinant DNA technology has seen exponential growth in both application and sophistication, yielding increasingly powerful tools for DNA manipulation. Cloning genes is now so simple and efficient that it has become a standard laboratory technique. This has led to an explosion in the understanding of gene function in recent decades. Emerging technologies promise even greater possibilities, such as enabling researchers to seamlessly stitch together multiple DNA fragments and transform the resulting plasmids into bacteria, in under two hours, or the use of swappable gene cassettes, which can be easily moved between different constructs, to maximize speed and flexibility. In the near future, molecular cloning will likely see the emergence of a new paradigm, 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.
Rebecca Tirabassi, Bitesize Bio.
Molecular cloning refers to the isolation of a DNA 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 to generate many copies of the DNA for analysis of the gene sequence, and/or to express the resulting protein for the study or utilization of the protein’s function. The clones can also be manipulated and mutated in vitro to alter the expression and function of the protein.The basic cloning workflow includes four steps:
- Isolation of target DNA fragments (often referred to as inserts)
- Ligation of inserts into an appropriate cloning vector, creating recombinant molecules (e.g., plasmids)
- Transformation of recombinant plasmids into bacteria or other suitable host for propagation
- Screening/selection of hosts containing the intended recombinant plasmid
These four ground-breaking steps were carefully pieced together and performed by multiple laboratories, beginning in the late 1960s and early 1970s. A summary of the discoveries that comprise traditional molecular cloning is described in the following pages.
The Foundation of Molecular Cloning
Cutting (Digestion). Recombinant DNA technology first emerged in the late 1960s, with the discovery of enzymes that could specifically cut and join double-stranded DNA molecules. In fact, as early as 1952, two groups independently observed that bacteria encoded a “restriction factor” that prevented bacteriophages from growing within certain hosts (1,2). But the nature of the factor was not discovered until 1968, when Arber and Linn succeeded in isolating an enzyme, termed a restriction factor, that selectively cut exogenous DNA, but not bacterial DNA (3). These studies also identified a methylase enzyme that protected the bacterial DNA from restriction enzymes.
Shortly after Arber and Linn’s discovery, Smith extended and confirmed these studies by isolating a restriction enzyme from Haemophilus influenza. He demonstrated that the enzyme selectively cut DNA in the middle of a specific 6 base-pair stretch of DNA; one characteristic of certain restriction enzymes is their propensity to cut the DNA substrate in or near specific, often palindromic, “recognition” sequences (4).
The full power of restriction enzymes was not realized until restriction enzymes and gel electrophoresis were used to map the Simian Virus 40 (SV40) genome (5). For these seminal findings, Werner Arber, Hamilton Smith, and Daniel Nathans shared the 1978 Nobel Prize in Medicine.
Assembling (Ligation). Much like the discovery of enzymes that cut DNA, the discovery of an enzyme that could join DNA was preceded by earlier, salient observations. In the early 1960s, two groups discovered that genetic recombination could occur though the breakage and ligation of DNA molecules (6,7), closely followed by the observation that linear bacteriophage DNA is rapidly converted to covalently closed circles after infection of the host (8). Just two years later, five groups independently isolated DNA ligases and demonstrated their ability to assemble two pieces of DNA (9-13).
Not long after the discovery of restriction enzymes and DNA ligases, the first recombinant DNA molecule was made. In 1972, Berg separately cut and ligated a piece of lambda bacteriophage DNA or the E. coli galactose operon with SV40 DNA to create the first recombinant DNA molecules (14). These studies pioneered the concept that, because of the universal nature of DNA, DNA from any species could be joined together. In 1980, Paul Berg shared the Nobel Prize in Chemistry with Walter Gilbert and Frederick Sanger (the developers of DNA sequencing), for “his fundamental studies of the biochemistry of nucleic acids, with particular regard to recombinant DNA.”
Transformation. Recombinant DNA technology would be severely limited, and molecular cloning impossible, without the means to propagate and isolate the newly constructed DNA molecule. The ability to transform bacteria, or induce the uptake, incorporation and expression of foreign genetic material, was first demonstrated by Griffith when he transformed a non-lethal strain of bacteria into a lethal strain by mixing the non-lethal strain with heat-inactivated lethal bacteria (15). However, the nature of the “transforming principle” that conveyed lethality was not understood until 1944. In the same year, Avery, Macleod and McCarty demonstrated that DNA, and not protein, was responsible for inducing the lethal phenotype (16).
Initially, it was believed that the common bacterial laboratory strain, E. coli, was refractory to transformation, until Mandel and Higa demonstrated that treatment of E. coli with calcium chloride induced the uptake of bacteriophage DNA (17). Cohen applied this principle, in 1972, when he pioneered the transformation of bacteria with plasmids to confer antibiotic resistance on the bacteria (18).
The ultimate experiment: digestion, ligation and transformation of a recombinant DNA molecule was executed by Boyer, Cohen and Chang in 1973, when they digested the plasmid pSC101 with EcoRI, ligated the linearized fragment to another enzyme-restricted plasmid and transformed the resulting recombinant molecule into E. coli, conferring tetracycline resistance on the bacteria (19), thus laying the foundation for most recombinant DNA work since.
Building on the Groundwork
While scientists had discovered and applied all of the basic principles for creating and propagating recombinant DNA in bacteria, the process was inefficient. Restriction enzyme preparations were unreliable due to non-standardized purification procedures, plasmids for cloning were cumbersome, difficult to work with and limited in number, and experiments were limited by the amount of insert DNA that could be isolated. Research over the next few decades led to improvements in the techniques and tools available for molecular cloning.
Early vector design.
Development of the first standardized vector. Scientists working in Boyer’s lab recognized the need for a general cloning plasmid, a compact plasmid with unique restriction sites for cloning in foreign DNA and the expression of antibiotic resistance genes for selection of transformed bacteria. In 1977, they described the first vector designed for cloning purposes, pBR322 (20). This vector was small, ~4 kilobases in size, and had two antibiotic resistance genes for selection.
Vectors with on-board screening and higher yields. Although antibiotic selection prevented non-transformed bacteria from growing, plasmids that re-ligated without insert DNA fragments (self-ligation) could still confer antibiotic resistance on bacteria. Therefore, finding the correct bacterial clones containing the desired recombinant DNA molecule could be time-consuming.
Vieira and Messing devised a screening tool to identify bacterial colonies containing plasmids with DNA inserts. Based upon the pBR322 plasmid, they created the series of pUC plasmids, which contained a “blue/white screening” system (21). Placement of a multiple cloning site (MCS) containing several unique restriction sites within the LacZ´ gene allowed researchers to screen for bacterial colonies containing plasmids with the foreign DNA insert. When bacteria were plated on the correct media, white colonies contained plasmids with inserts, while blue colonies contained plasmids with no inserts. pUC plasmids had an additional advantage over existing vectors; they contained a mutation that resulted in higher copy numbers, therefore increasing plasmid yields.
Improving restriction digests. Early work with restriction enzymes was hampered by the purity of the enzyme preparation and a lack of understanding of the buffer requirements for each enzyme. In 1975, New England Biolabs (NEB) became the first company to commercialize restriction enzymes 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, have been discovered by scientists across the globe [for a complete list of restriction enzymes and recognition sequences, visit REBASE® at rebase.neb.com (22)]. NEB currently supplies over 230 of these specificities.
NEB was also one of the first companies to develop a standardized four-buffer system, and to characterize all of its enzyme activities in this buffer system. This led to a better understanding of how to conduct a double digest, or the digestion of DNA with two enzymes sim-ultaneously. Later research led to the development of one-buffer systems, which are compatible with the most common restriction enzymes (such as NEB’s CutSmart™ Buffer).
With the advent of commercially available restriction enzyme libraries with known sequence specificities, restriction enzymes became a powerful tool for screening potential recombinant DNA clones. The “diagnostic digest” was, and still is, one of the most common techniques used in molecular cloning.
Vector and insert preparation. Cloning efficiency and versatility were also improved by the development of different techniques for preparing vectors prior to ligation. Alkaline phosphatases were isolated that could remove the 3´ and 5´ phosphate groups from the ends of DNA [and RNA; (23)]. It was soon discovered that treatment of vectors with Calf-Intestinal Phosphatase (CIP) dephosphorylated DNA ends and prevented self-ligation of the vector, increasing recovery of plasmids with insert (24).
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-labile alkaline phosphatases, such as recombinant Shrimp Alkaline Phosphatase (rSAP) and Antarctic Phosphatase (AP) (both sold by NEB), decreased the steps and time involved, as a simple shift in temperature inactivates the enzyme prior to the ligation step (25).
DNA sequencing arrives. DNA sequencing was developed in the late 1970s when two competing methods were devised. Maxam and Gilbert developed the “chemical sequencing method,” which relied on chemical modification of DNA and subsequent cleavage at specific bases (26). At the same time, Sanger and colleagues published on the “chain-termination method”, which became the method used by most researchers (27). The Sanger method quickly became automated, and the first automatic sequencers were sold in 1987.
The ability to determine the sequence of a stretch of DNA enhanced the reliability and versatility of molecular cloning. Once cloned, scientists could sequence clones to definitively identify the correct recombinant molecule, identify new genes or mutations in genes, and easily design oligonucleotides based on the known sequence for additional experiments.
The impact of the polymerase chain reaction. One of the problems in molecular cloning in the early years was obtaining enough insert DNA to clone into the vector. In 1983, Mullis devised a technique that solved this problem and revolutionized molecular cloning (28). He amplified a stretch of target DNA by using opposing primers to amplify both complementary strands of DNA, simultaneously. Through cycles of denaturation, annealing and polymerization, he showed he could exponentially amplify a single copy of DNA. The polymerase chain reaction, or PCR, made it possible to amplify and clone genes from previously inadequate quantities of DNA. For this discovery, Kary Mullis shared the 1993 Nobel Prize in Chemistry “for contributions to the developments of methods within DNA-based chemistry”.
In 1970, Temin and Baltimore independently discovered reverse transcriptase in viruses, an enzyme that converts RNA into DNA (29,30). Shortly after PCR was developed, reverse transcription was coupled with PCR (RT-PCR) to allow cloning of messenger RNA (mRNA). Reverse transcription was used to create a DNA copy (cDNA) of mRNA that was subsequently amplified by PCR to create an insert for ligation. For their discovery of the enzyme, Howard Temin and David Baltimore were awarded the 1975 Nobel Prize in Medicine and Physiology, which they shared with Renato Dulbecco.
Cloning of PCR products. The advent of PCR meant that researchers could now clone genes and DNA segments with limited knowledge of amplicon sequence. However, there was little consensus as to the optimal method of PCR product preparation for efficient ligation into cloning vectors.
Several different methods were initially used for cloning PCR products. The simplest, and still the most common, method for cloning PCR products is through the introduction of restriction sites onto the ends of the PCR product (31). This allows for direct, directional cloning of the insert into the vector after restriction digestion. Blunt-ended cloning was developed to directly ligate PCR products generated by polymerases that produced blunt ends, or inserts engineered to have restriction sites that left blunt ends once the insert was digested. This was useful in cloning DNA fragments that did not contain restriction sites compatible with the vector (32).
Shortly after the introduction of PCR, overlap extension PCR was introduced as a method to assemble PCR products into one contiguous DNA sequence (33). In this method, the DNA insert is amplified by PCR using primers that generate a PCR product containing overlapping regions with the vector. The vector and insert are then mixed, denatured and annealed, allowing hybridization of the insert to the vector. A second round of PCR generates recombinant DNA molecules of insert-containing vector. Overlap extension PCR enabled researchers to piece together large genes that could not easily be amplified by traditional PCR methods. Overlap extension PCR was also used to introduce mutations into gene sequences (34).
Development of specialized cloning techniques.
In an effort to further improve the efficiency of molecular cloning, several specialized tools and techniques were developed that exploited the properties of unique enzymes.
TA Cloning. One approach took advantage of a property of Taq DNA Polymerase, the first heat-stable polymerase used for PCR. During amplification, Taq adds a single 3´ dA nucleotide to the end of each PCR product. The PCR product can be easily ligated into a vector that has been cut and engineered to contain single T residues on each strand. Several companies have marketed the technique and sell kits containing cloning vectors that are already linearized and “tailed”.
LIC. Ligation independent cloning (LIC), as its name implies, allows for the joining of DNA molecules in the absence of DNA ligase. LIC is commonly performed with T4 DNA Polymerase, which is used to generate single-stranded DNA overhangs, >12 nucleotides long, onto both the linearized vector DNA and the insert to be cloned (35). When mixed together, the vector and insert anneal through the long stretch of compatible ends. The length of the compatible ends is sufficient to hold the molecule together in the absence of ligase, even during transformation. Once transformed, the gaps are repaired in vivo. There are several different commercially available products for LIC. USER cloning.
USER cloning was first developed in the early 1990s as a restriction enzyme- and ligase-independent cloning method (36). When first conceived, the method relied on using PCR primers that contained a ~12 nucleotide 5´ tail, in which at least four deoxythymidine bases had been substituted with deoxyuridines. The PCR product was treated with uracil DNA glycosidase (UDG) and Endonuclease VIII, which excises the uracil bases and leaves a 3´ overlap that can be annealed to a similarly treated vector. NEB sells the USER enzyme for ligase and restriction enzyme independent cloning reactions.
Molecular cloning has progressed from the cloning of a single DNA fragment to the assembly of multiple DNA components into a single contiguous stretch of DNA. New and emerging technologies seek to transform cloning into a process that is as simple as arranging “blocks” of DNA next to each other.
DNA assembly methods. Many new, elegant technologies allow for the assembly of multiple DNA fragments in a one-tube reaction. The advantages of these technologies are that they are standardized, seamless and mostly sequence independent. In addition, the ability to assemble multiple DNA fragments in one tube turns a series of previously independent restriction/ligation reactions into a streamlined, efficient procedure.
Different techniques and products for gene assembly include SLIC (Sequence and Ligase Independent Cloning), Gibson Assembly (NEB), GeneArt® Seamless Cloning (Life Technologies) and Gateway® Cloning (Invitrogen) (35,37,38).
In DNA assembly, blocks of DNA to be assembled are PCR amplified. Then, the DNA fragments to be assembled adjacent to one another are engineered to contain blocks of complementary sequences that will be ligated together. These could be compatible cohesive ends, such as those used for Gibson Assembly, or regions containing recognition sites for site-specific recombinases (Gateway). The enzyme used for DNA ligation will recognize and assemble each set of compatible regions, creating a single, contiguous DNA molecule in one reaction.
Synthetic biology. DNA synthesis is an area of synthetic biology that is currently revolutionizing recombinant DNA technology. Although a complete gene was first synthesized in vitro in 1972 (40), DNA synthesis of large DNA molecules did not become a reality until the early 2000s, when researchers began synthesizing whole genomes in vitro (41,42). These early experiments took years to complete, but technology is accelerating the ability to synthesize large DNA molecules.
In the last 40 years, molecular cloning has progressed from arduously isolating and piecing together two pieces of DNA, followed by intensive screening of potential clones, to seamlessly assembling up to 10 DNA fragments with remarkable efficiency in just a few hours, or designing DNA molecules in silico and synthesizing them in vitro. Together, all of these technologies give molecular biologists an astonishingly powerful toolbox for exploring, manipulating and harnessing DNA, that will further broaden the horizons of science. Among the possibilities are the development of safer recombinant proteins for the treatment of diseases, enhancement of gene therapy (43), and quicker production, validation and release of new vaccines (44). But ultimately, the potential is constrained only by our imaginations.
Rebecca Tirabassi is an Assistant Editor at Bitesizebio.com.
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