PCR Based Cloning Kits: Something For Everybody

Date: April 13, 1998PCR Based Cloning Kits Table The End Table (PDF Format) PCR has found applications in almost every imaginable facet of molecular biology, and for many applications, looking at a band on a gel is not enough. Sequencing, expressing, mutating--all require cloning. And as it happens, cloning strategies that work for other types of DNA fragments don't work at all well, or require inordinate effort, with PCR fragments. For example, the most commonly used cloning strategy requires that the insert and vector have compatible ends. Thus, in order to clone PCR fragments by this technique, restriction enzyme recognition sites have to be engineered into the PCR primers, adding to the time required and cost of cloning. Another common cloning strategy employed, blunt-end ligation, particularly useful when working with incompatible ends, is inefficient when applied to PCR fragments because many polymerases used in PCR, as it happens, add additional nucleotides to the ends of the fragments, creating staggered ends. PCR-based cloning kits have come along to fill this niche. Novel approaches to cloning PCR fragments have been devised, and as detailed in the company profiles and the table that follows, while sharing some properties, each kit is unique either in approach or in the features offered. Capitalizing on the ragged ends created by some thermophilic polymerases, in particular the 3´A overhang left by Taq polymerase, the first generation of PCR-based cloning kits used vectors with compatible overhangs. These so-called TA vectors, still among the offerings by CLONTECH, Invitrogen, Genpak, and Promega, employ vectors with T overhangs to complement PCR fragments with A overhangs. As other thermophilic enzymes with specific and different extendase activities--that is, they leave different ends (see The End Table)--came into use, other approaches needed to be devised. Blunting the ends of PCR fragments (or polishing them, as some companies prefer to call it) is a co mon strategy. A DNA polymerase like Klenow can be used to fill in the ends, or alternatively, Pfu DNA polymerase can be used to remove extended bases with its 3´ to 5´ exonuclease activity. One useful feature of Pfu is that since it is active only at high temperatures (>65°C), it does not need to be removed prior to ligation, which is typically done at or below room temperature. On the other hand, blunt ended-cloning is notoriously inefficient, with very high vector backgrounds commonly as high as 80 percent. Several companies have devised cloning systems that tackle this problem. Positive selection (in which only colonies containing plasmids with inserts survive, as compared with blue/white screening in which colonies need only have a plasmid) is available in three PCR-based cloning systems. The GenHunter vector is designed so that the insert interrupts a lac I repressor gene, which when intact represses the gene for tetracycline resistance. Hence, when plating on tet-containing media, only cells with insert-containing vectors will survive, through the release of the repression on the tet gene. Invitrogen's ZeroBlunt Vector and Quantum Biotechnologies' QuanTox carry a gene for a toxic peptide into which inserts are cloned, so that, again, only recombinants survive. Nonrecombinants produce the toxic peptide and are killed. Stratagene has taken a different approach to improving blunt-end ligation efficiencies with its PCR-Script Cloning System, in which a restriction enzyme is included in the ligation reaction. Having restriction enzyme present during ligation keeps linearized vector in solution by re-restricting plasmids as they ligate intramolecularly. (Intermolecular ligation--vector to insert--does not create the endonuclease target site, and hence recombinant molecules are not subject to the same fate.) This enzyme combination reduces vector background and hence improves ligation efficiency of blunt-ended DNA fragment. Eliminating the ligation step altogether, and providing for directional cloning at the same time, are ligation- independent cloning (LIC) kits offered by Novagen and Pharmingen. LIC vectors are constructed to have noncomplementary 13- and 14-base single-stranded overhangs, produced by incubating the vector with T4 DNA polymerase in the presence of a single dNTP. The 3´* 5´ prime exonuclease activity of the polymerase chews at the vector until it reaches the nucleotide included in the reaction, at which point the exonuclease stops. When properly engineered, this leads to a vector with unique, non-complementary overhangs. PCR fragments with compatible ends can be made using primers with the appropriate 5´ extensions. The likelihood of vector reannealing is greatly reduced since the ends are not complementary, which simplifies cloning since the production of recombinant species is less likely to be left to chance. The annealed LIC vector and insert can then be transformed directly into competent E. coli cells, where the cellular machinery takes care of making the covalent bond between vector and insert regions. Rather than engineering a vector for accepting PCR fragments, two companies have taken the opposite tack and instead prepare the amplified fragments for insertion into any vector. The Amersham Pharmacia kit prepares PCR fragments for insertion into any blunt-ended vector, while the Sigma kit creates EcoRI ends on PCR fragments, compatible with any EcoRI-digested vector. Most vectors have incorporated some regulatory regions to facilitate various downstream applications of the PCR fragments: Sequencing primers: most vectors have either the f1 origin of replication for making single-stranded template or M13 primer sites for direct sequencing. Promoters: RNA polymerase promoters active in bacterial cells have been incorporated into vectors for making probes or for in vitro transcription studies. Transcription and translation regulatory regions: prokaryotic (Novagen), eukaryotic (Invitrogen, Pharmingen, Promega, and Stratagene) and baculovirus (Novagen and Pharmingen) regulatory elements have been incorporated to facilitate the expression of PCR-amplified genes. Kits also come with a variety of accoutrements: some kits supply columns for purifying the PCR fragments prior to cloning; some come with competent cells; some provide special primers for cloning and/or sequencing; and most come with some kind of positive control system. 5 Prime »3 Prime's PRIME PCR CLONER™ cloning system can be used with all PCR products, those containing A overhangs or blunt-ended. The kit comes in two versions that differ only in the clean-up system provided: PCR Select II is preferred for inserts greater than 100 base pairs, and the PCR Select III is for inserts greater than 300 base pairs. Both kits come with a precut, dephosphorylated pNoTA/T7 vector, which contains the T7 polymerase promoter, the lacZ-alpha gene into which the insert is cloned, and the ampicillin-resistance gene. After PCR, the products are purified on spin columns (provided) to remove salts, unincorporated nucleotides, and primer dimers prior to cloning; incubated with the modification reagent, which blunts the ends of the PCR fragments; and then followed by a 30-minute ligation reaction. Sequencing can be performed directly on the constructs using the M13/pUC universal primer. The T7 promoter can be used both for in vitro production of run-off transcripts as well as in vivo for gene expression of cloned inserts in certain E. coli host bacteria. Subcloning is facilitated by the presence of single and double restriction sites flanking the insert site on the plasmid. The kit comes with vector, purification spin columns (20), proprietary cloning reagent, proprietary modification reagent, ligation buffer, nucleotide solution, competent cells, X-Gal, IPTG, and a blue/white positive kit control. The SureClone Ligation Kit from Amersham Pharmacia Biotech is intended for cloning PCR products directly into any vector; the kit can be used with most blunt-ended, double-stranded vectors. PCR products are first subjected to a 30 minute blunting/kinasing reaction to remove overhangs from the PCR products and to phosphorylate the ends, followed by spin column purification (column provided). Ligation follows into a cloning vector, such as pUC18, which comes SmaI digested (blunt-ended) and dephosphorylated. The kit comes with FPLCpure Klenow fragment, FPLCpure T4 polynucleotide kinase, FPLCpure T4 DNA ligase, Sephacryl S-200 HR, and microspin columns, buffers, and control PCR mix. The AdvanTAge(TM) PCR Cloning Kit is designed for direct T/A cloning of PCR products without requiring special primers, post-PCR purification, or enzymatic treatment. The method involves ligating PCR products freshly made with Taq (and its derivatives) or Tth polymerases into the pTAdv vector, supplied precut and ready for ligation. The kit can be used to clone and analyze PCR products from a number of CLONTECH kits, including the PCR Select™ cDNA Subtraction Kits, Marathon™ cDNA Amplification Kit, and Genome Walker, a PCR-based genomic cloning kit. Kit components include vector, control DNA template and primers, T4 DNA ligase, and competent cells. The PCR-TRAP® Cloning System is a positive-selection system for cloning PCR products through blunt-end ligation with up to 90 percent efficiency (90 percent of the transformants bear recombinant plasmids). The PCR products require neither post-PCR purification nor modifications. This system is unique in that it utilizes the lambda repressor to provide a positive-selection mechanism for insert- containing transformants. The vector is designed such that the cloning site is embedded in the lamdba repressor gene, cI, which is engineered to repress the promoter for the tetracycline-resistance gene. Cloning into the cI gene leads to the inactivation of the repressor gene, thus turning on the tet-resistance gene. The vector is designed with sequences flanking the insert that can be used for the preparation of probes (primers included) or for sequencing (primers included). Genpak's pTT Cloning System allows cloning of PCR fragments by ligating the 3´ overhangs created by Taq polymerase to complementary T overhangs on the pTT vector. The pTT vector, based on M13, contains a polylinker with 11 unique restriction sites, the lacZ gene for blue/white selection, the ampicillin genes for antibiotic selection, and the f1 origin of replication for the production of single-stranded DNA. The TCL20 kit comes with enough vector, ligase, and buffer for 20 reactions and DH alpha-F´ competent cells; the TL20 kit comes with vector, ligase, and buffer for 20 reactions; and the T20 kit comes with 1µg of pTT vector alone. Ligation is carried out with purified PCR fragments and vector for 3 hours at 4°C, and transformation for the usual 75 minutes. Colonies can be screened for inserts either by performing PCR on cultures using M13+/- primers or by picking white colonies and testing them for inserts. Single-stranded DNA can be prepared by overnight culturing with helper phage. Invitrogen offers a range of kits for the direct cloning of PCR products: Original TA Cloning® Kit; Unidirectional and Bidirectional Eukaryotic TA Cloning® Kits; and ZeroBlunt (TM) PCR Cloning Kit. All of Invitrogen's TA Cloning® vectors are supplied linearized with single 3´T overhangs for direct ligation of PCR products with 3´A overhangs. TA cloning eliminates enzymatic blunting, gel or column purification of PCR products, and prior knowledge of PCR product sequence. The ZeroBlunt vector uses positive selection via disruption of the highly lethal gene ccdB. Without an insert, ZeroBlunt expresses the ccdB protein, which results in cell death, eliminating vector background. When a fragment is inserted, ccd B expression is disrupted and recombinants are able to grow. Invitrogen's latest innovation in the field of PCR based cloning is the TOPO-Cloning (TM) technology, which offers five-minute cloning of PCR products. TOPO-Cloning uses novel cloning vectors that are preactivated with topoisomerase-I. Topoisomerase-I possesses rapid ligation activity when combined with the appropriate vector DNA. When an insert with compatible overhang is added, the topoisomerse-activated vector ligates it in five minutes at room temperature. The TOPO TA Cloning Kit is based on the TA Cloning method and uses the pCR2.1-TOPO vector. Other TOPO cloning kits are available for cloning Expand-, eLONGase- and other polymerase generated-PCR products, and for the expression of recombinant proteins in E. coli (pBAD/V5- His TOPO), Drosophila S2 (pMT/V5-His TOPO), and mammalian cells (pcDNA3.1/V5-His TOPO). Each expression kit comes with an expression control, TOPO vector, sequencing primers, OneShot Competent cells, PCR reagents (except a thermostable polymerase), and controls. Novagen offers a variety of kits in one of two basic approaches. Perfectly Blunt (TM) Cloning Kits are designed for cloning of any DNA fragment, regardless of whether the termini are blunt, have 5´ overhangs, or 3´ overhangs. Target DNA is first converted to a blunt, phosphorylated form; ligated into the blunt-ended, dephosphorylated vector; and transformed into NovaBlue cells. Insert-containing clones are distinguished using blue/white screening. The pT7 vectors are designed for use as a general purpose cloning and sequencing vector (pT7Blue) or for general purpose cloning and sequencing with enhanced in vitro translation capabilities (pT7Blue-2). In addition, vectors with an extensive multiple cloning region (pT7 Blue-3) and with dual promoters (pST BlueBlunt Vector) are also available. Novagen also offers a selection of LIC kits, developed for the directional cloning and expression of PCR products, without restriction enzyme digestion or ligation reactions. The LIC vectors have an enterokinase (Ek/LIC) or factor Xa (Xa/LIC) cleavage site adjacent to the insert site so that vector-encoded fusion sequences can be removed from expressed proteins. A series of vectors, the pET-LIC Vectors are designed for cloning and high level expression of cloned sequence in E. coli, with tags and fusions for detection and purification of target proteins--thioredoxin, His-Tag®, S·Tag (TM) , and CBD™ Tag. A second series of vectors with tags and fusion sequences, pBAC (TM)/LIC, is offered for expressing cloned sequences in the baculovirus system. Finally, the pCITE®-5Ek/LIC Vector includes a sequence that increases the translation efficiency of adjacent sequences in rabbit reticulocyte lysates. The LA PCR in vitro Cloning Kit is designed specifically to amplify unknown regions of DNA. This kit takes advantage of LA PCR technology by including the TaKaRa LA Taq enzyme and LA Buffer II for longer amplification with high fidelity (reduced occurrence of mutation when cloning). Amplification is accomplished by cassette-mediated PCR: target DNA is first digested with a restriction enzyme, after which an oligo containing a restriction site is ligated to the 3´-end of a restriction fragment containing a region of known DNA sequence. The first round of PCR is performed between this primer and a specific primer (user supplied) to amplify an unknown region. The long strand synthesized can then be used as a template for subsequent rounds of amplification. This allows long DNA fragments, such as those found in genomic DNA, to be obtained without the need for time-consuming library construction and screening. PharMingen offers three LIC cloning kits, enabling the cloning of PCR fragments without enzymatic digestion or knowledge of the sequence of the PCR insert. The pPMG-LIC Bacterial Cloning Kit has a LIC site surrounded by SP6 and T7 polymerase promoters for in vitro transcription studies, f1 origin of replication for the generation of single-stranded templates, and several unique cloning sites to facilitate the retrieval of inserts. The pCMV-LIC Mammalian Expression Kit, which has the LIC site downstream from the CMV promoter, is designed for high level expression of cloned genes in cultured mammalian cells. The vector also has an SV40 origin of replication, human growth hormone regulatory sequences for directing RNA processing, f1 origin of replication, and ampicillin resistance. The pAcSG2-LIC Baculovirus Expression Kit, which contains the polyhedrosis virus promoter upstream from the LIC cloning site, can be used to generate recombinant baculovirus particles. All PharMingen LIC kits require that the insert DNA be amplified with primers (not provided) that have been specifically designed with 13 additional nucleotides incorporated at the 5´-ends. The length and GC content of the tails are sufficient to create stable annealing of the vector and PCR product within 30 minutes. The annealed construct can be used to transform bacteria without ligation. All LIC kits contain pretreated DNA polymerase vector, dTTP's, T4 DNA polymerase, T4 DNA polymerase buffer, and control insert. The pGEM®-T and pGEM®-T Easy Vector Systems are designed for cloning fragments with 3´A overhangs. The vectors were prepared by cutting Promega's pGEM®-5Zf(+) Vectors with EcoR V and adding a 3´-terminal thymidine to both ends. Since some thermostable DNA polymerases used in PCR result in blunt-end fragments, a simple modification is required for blunt-end PCR products prior to cloning into pGEM®-T and pGEM®-T Easy Vectors. The pGEM®-T and pGEM®-T Easy Vectors contain T7 and SP6 RNA polymerase promoters flanking a multiple cloning site (MCS) within the ß-galactosidase gene for direct identification of insert-containing colonies by color screening. Both the pGEM®-T and pGEM®-T Easy Vector contain multiple restriction sites within the MCS, allowing for the release of the insert by digestion with a single restriction enzyme. They also contain the origin of replication of the filamentous phage f1 for the preparation of single-stranded DNA. The pTargeT Mammalian Expression Vector System, derived from Promega's pCI-neo Mammalian Expression Vector, contains the ß-galactosidase gene, the human cytomegalovirus (CMV) immediate-early enhancer/ promoter region to promote constitutive expression of cloned DNA inserts in mammalian cells, and the neomycin phosphotransferase gene, a selectable marker for mammalian cells. The vector can be used for transient expression or for stable expression by selecting transfected cells with the antibiotic G-418. QuanTox™ and QuanTox-Blunt™ are general blunt-end cloning vectors containing a gene coding for a toxic peptide that inhibits bacterial growth when expression is induced. Ligation of a DNA fragment into the multiple cloning site present in these vectors disrupts the gene coding for the toxic peptide. The recombinant molecules are thennontoxic, while the vectors in the absence of insert produce a toxic peptide. This constitutes a "positive selection system" for cohesive or blunt-ended recombinant molecules. The ExoClone (TM) PCR Cloning Kit provides a process for cloning any PCR product, up to 6 kb, into any vector with an EcoR I site in its multiple cloning site. ExoClone generates amplicons that can be ligated to EcoR I digested vectors. The kit takes advantage of the fact that phosphorothioate nucleotides are resistant to cleavage by exonuclease III. Primers are designed to include the additional deoxynucleotides P-AATTC on the 5´ end. Following amplification in the presence of dATP, dCTP, TTP and thio-dGTP, the fragment is treated with exonuclease III, which degrades in the 3´»5´ direction. The degradation stops when the enzyme reaches the thioester, leaving a 4-base overhang identical to an EcoR I digestion product. These sticky ends allow the ligation of the PCR product into any vector containing an EcoR I site. Because EcoR I is not used to generate the sticky ends, there is no need for the amplified product to be devoid of EcoR I sites. Cloning efficiencies of 1 x 108 and greater have been routinely achieved. Stratagene's PCR-Script™ Cloning Kits allow the cloning of all PCR products, regardless of the PCR enzyme used in the reaction, though they work best with blunt-ended PCR products such as those made with Pfu DNA polymerase. Products from low fidelity polymerases, like Taq polymerase, can be efficiently cloned into PCR-Script cloning vectors after the ends are polished with reagents provided in the kits. PCR products are incubated with one of the predigested PCR-Script cloning vectors, Srf I and T4 DNA ligase. Using the restriction enzyme in the ligation reaction maintains a high steady-state concentration of digested vector DNA and allows the use of nonphosphorylated, unmodified PCR primers. Ligation efficiency of blunt-ended DNA fragments is increased by the simultaneous, opposing reactivity of the Srf I enzyme and the T4 DNA ligase on nonrecombinant vector DNA. PCR-Script Cloning Kits come with either ampicillin (PCR-Script PCR Amp Cloning Kit) or chloramphenicol (PCR-Script PCR Cam Cloning Kit) resistance markers. The pCMV-Script™ PCR Cloning Kit is designed for high level constitutive expression in mammalian cells, using the CMV promoter and the SV40 polyadenylation site. In addition, the neomycin- resistance gene is under control of the prokaryotic ß-lactamase promoter to provide kanamycin resistance in bacteria as well as the SV40 early promoter to provide G418 resistance in mammalian cells. Each kit comes with vector, restriction enzyme, ligase, dNTPs, Pfu polymerase and polishing buffer, XL-10 Gold™ Ultra competent cells, the StrataPrep PCR purification kit, and a control plasmid and insert. PCR Based Cloning Kits Table The End Table (PDF Format) The author can be reached at ldefrancesco@the-scientist.com

PCR Based Cloning Kits: Something For Everybody

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