FastCloning: a highly simplified, purification-free, sequence- and ligation-independent PCR cloning method
© Li et al; licensee BioMed Central Ltd. 2011
Received: 3 June 2011
Accepted: 12 October 2011
Published: 12 October 2011
Although a variety of methods and expensive kits are available, molecular cloning can be a time-consuming and frustrating process.
Here we report a highly simplified, reliable, and efficient PCR-based cloning technique to insert any DNA fragment into a plasmid vector or into a gene (cDNA) in a vector at any desired position. With this method, the vector and insert are PCR amplified separately, with only 18 cycles, using a high fidelity DNA polymerase. The amplified insert has the ends with ~16-base overlapping with the ends of the amplified vector. After DpnI digestion of the mixture of the amplified vector and insert to eliminate the DNA templates used in PCR reactions, the mixture is directly transformed into competent E. coli cells to obtain the desired clones. This technique has many advantages over other cloning methods. First, it does not need gel purification of the PCR product or linearized vector. Second, there is no need of any cloning kit or specialized enzyme for cloning. Furthermore, with reduced number of PCR cycles, it also decreases the chance of random mutations. In addition, this method is highly effective and reproducible. Finally, since this cloning method is also sequence independent, we demonstrated that it can be used for chimera construction, insertion, and multiple mutations spanning a stretch of DNA up to 120 bp.
Our FastCloning technique provides a very simple, effective, reliable, and versatile tool for molecular cloning, chimera construction, insertion of any DNA sequences of interest and also for multiple mutations in a short stretch of a cDNA.
Molecular cloning is one of the most widely used techniques in biomedical research laboratories. Traditionally, molecular cloning joins insert and vector by T4 DNA ligase after restriction digestion to excise insert from a donor vector or from a PCR product with restriction enzyme recognition sites added to the ends . Although this is a widely used method, it involves multiple steps and is time consuming. This multi-step process also makes it difficult or complicated for troubleshooting. To overcome the difficulties encountered in the original cloning method, many other alternative cloning methods have been developed over the last two decades. These methods include TA cloning , ligation independent cloning with T4 DNA polymerase [3, 4], GATEWAY recombinational cloning , and more recent sequence- and ligation-independent cloning kits, such as CloneEZ (GenScript USA Inc., Piscataway, NJ, USA), one step cloning , and overlap extension PCR cloning . However, each of these techniques has its own limitations. For example, TA cloning uses regular Taq DNA polymerase to add a single 3'-A overhang to the ends of the PCR product. The PCR product is directly cloned into a TA cloning vector with a complementary 3'-T overhang in both ends without restriction digestion. The limitations of this method are low fidelity of Taq DNA polymerase causing unwanted mutations and requirement of subcloning into the final target vector with restriction digestion and ligation. The early ligation independent cloning uses the 3'-exonucnease activity of T4 DNA polymerase to create 15-base 5'overhangs in the ends of insert and complementary 5' overhangs in the ends of vector. This technique requires specific sequences to create 15-base overhangs. Gateway recombinational cloning uses site-specific recombination to transfer cDNAs between donor and destination vectors, which requires additional specific sequences for recombination. The latest ligation-independent cloning, such as CloneEZ and In-Fusion cloning kits, uses some DNA polymerase to generate sticky ends in the vector and insert without specific sequence requirement, except for restriction sites to linearize the vector. However, the new ligation independent cloning still requires purification of the digested vector and PCR-amplified insert, and the purchase of purification and cloning kits. Similarly, overlap extension PCR cloning also requires purification of the first round PCR products (vector and insert) and an additional round overlap extension PCR, which usually generates multiple bands, for producing linked vector and insert. One-step "quick assemble" cloning does not need purification of PCR products. However, it includes two sequential 35-cycle PCRs with a total number of 70 cycles. The first-round PCRs are used to amplify insert and linear vector. The second-round PCR is essentially the overlap extension PCR to assemble vector and insert into a single linear PCR product. Another ligation independent cloning technique, using nick DNA endonuclease to create long single-strand 5' overhangs in the vector and PCR-amplified insert , requires specific sequences for nick DNA endonuclease and purification of the PCR product.
Purification of PCR product not only takes extra time and requires purification kit, but also potentially creates additional problems. For example, to compensate for the loss of PCR product during purification, the number of PCR cycles is generally 25-30. High number of PCR cycles increases the chance of random mutations or, in our experiences, dramatically decreases cloning efficiency for the PCR products generated by high fidelity DNA polymerases. This is especially true for the enzyme with high processability, such as pfuUltraII DNA polymerase. The decrease in cloning efficiency cannot be completely overcome by using state-of-the-art cloning kits, such as CloneEZ. Using this kit (with gel purification of digested vectors and PCR-amplified inserts), the successful rate in our laboratory still varies significantly with overall successful rate less than 50%. In the PCR-based QuickChange mutagenesis, the number of PCR cycles is recommended to be 12-18. Increase in PCR cycles could decrease efficiency according to the QuickChange protocol. Thus, these phenomena made us believe that the proofreading PCR enzymes could potentially damage the ends of PCR products if the number of PCR cycles increases. This could be due to the fact that the high fidelity DNA polymerases with the proofreading ability have 3' exonuclease activity. In the presence of dNTPs, the ends of PCR products can be protected from this 3' exonuclease activity. It is possible that depletion of dNTPs in high number of PCR cycles or dilution of dNTPs in the early stage of purification would weaken the 3' ends protection against 3' exonuclease activity of the DNA polymerase, which results in the damage of PCR products and makes cloning extremely difficult.
To circumvent the above-mentioned problems, we developed a cloning method termed FastCloning. With this method, both insert and vector are amplified by 18 PCR cycles with a high fidelity DNA polymerase. The unpurified PCR products of the vector and insert are then directly mixed at some ratios (see below) and digested by DpnI restriction enzyme to destroy their methylated DNA templates. Finally, the digested mixture is transformed into competent cells to obtain the target clones. The PCR amplification of vector is designed to make it possible to clone an insert into any position of the vector, to bypass vector digestion (and restriction site limitation) and purification steps, and to be compatible with DpnI digestion of the insert without further inactivation of the enzyme. Thus, this method can be used to construct cDNAs of fusion proteins or chimera without limitation by available restriction sites. In this study, we experimentally validated all these applications with our new method. In addition, a similar method can be easily adapted for deletion of a DNA fragment.
Primers used in the cloning or mutagenesis experiments
Reverse primer for pGEMHE vector with start codon
Forward primer for pGEMHE vector with stop codon
Reverse primer for p3xFlag-cmv-14 vector with start codon
Forward primer for p3xFlag-cmv-14 vector
Forward primer for CHRNA9 cloning into pGEMHE
Reverse primer for CHRNA9 cloning into pGEMHE
Forward primer for CHRNB2 cloning into pGEMHE
Reverse primer for CHRNB2 cloning into pGEMHE
Forward primer for CHRNA4 cloning into pGEMHE
Reverse primer for CHRNA4 cloning into pGEMHE
Forward primer for ECSCR cloning into pGEMHE
Reverse primer for ECSCR cloning into pGEMHE
Forward primer for ECSCR cloning into p3xFlag-cmv-14
Reverse primer for ECSCR cloning into p3xFlag-cmv-14
Forward primer for ZACN cloning into pGEMHE
Reverse primer for ZACN cloning into pGEMHE
Forward primer for APBB1 cloning into pGEMHE
Reverse primer for APBB1 cloning into pGEMHE
Forward primer of HTR3A for 5 Pro mutated to Ala
Reverse primer of HTR3A for 5 Pro mutated to Ala
Forward primer for pLXSN vector
Forward primer for pLXSN vector
Forward primer for Akt3v1 & v2 with Kozak sequence
Reverse primer for Akt3v1 cloning into pLXSN
Reverse primer for Akt3v2 cloning into pLXSN
Reverse primer for vector along with N-terminal cDNA
Forward primer for insert amplification
Reverse primer for C-terminal β2 insert amplification
Reverse primer for C-terminal β4 insert amplification
Forward primer for AChBP insert1 to α7 nAChR
Reverse primer for AChBP insert1 to α7 nAChR
Forward primer for AChBP insert2 to α7 nAChR
Reverse primer for AChBP insert2 to α7 nAChR
Forward primer for AChBP insert3 to α7 nAChR
Reverse primer for AChBP insert3 to α7 nAChR
Forward primer for AChBP insert4 to α7 nAChR
Reverse primer for AChBP insert4 to α7 nAChR
Forward primer for AChBP insert5 to α7 nAChR
Reverse primer for AChBP insert5 to α7 nAChR
The PCR reaction components were: 50 μl total volume, 0.5 μl Phusion DNA polymerase (New England Biolabs, Ipswich, MA), or 0.8 μl Pfu Turbo, or PfuUltra DNA polymerase (Agilent Technologies, Inc, Santa Clara, CA), 5 μl 10× buffer; 5 μl of 2.5 mM dNTPs; 10 ng of plasmid DNA template; 5 pmol of each primer. The PCR cycling parameters were 98°C 3 min, (98°C 10 sec, 55°C 30 sec, 72°C 20 sec/kb) × 18 cycles, 72°C 5 min, and 4°C infinite for Phusion DNA polymerase, and 95°C 3 min, (95°C 15 sec, 55°C 1 min, 72°C 1 min/kb) × 18 cycles, 72°C 5 min, and 4°C infinite for Pfu Turbo or PfuUltra DNA Polymerase. The PCR products (5 μl for each product) were examined with 1% agarose gel electrophoresis with ethidium bromide staining using VWR Mini Gel electrophoresis setup (VWR International, Marietta, GA, USA) running at 100 V for 30 min. The PCR products were then visualized under a UV transilluminator, and gel pictures were taken using an AGFA scanner.
After confirmation of PCR products, 1 μl of DpnI enzyme (New England Biolabs) was added into the remaining unpurified PCR reactions (45 μl for each product) for vector or insert separately. The vector and insert were then mixed with 1:1 ratio (1:1, 1:2, and 1:4 for α9 nAChR subunit), and digested at 37°C for 1 hour. Two micro-liters (2, 4, and 8 μl for α9 nAChR) of the digested vector-insert mixture were then added to 40 μl of chemically competent XL-10 Gold E. coli cells (prepared with rubidium chloride method) unless indicated otherwise. The mixture was then incubated for 30 min on ice. After heat shock at 42°C for 45 sec, 350 μl of SOC medium was added to the mixture. After 60 min shaking at 37°C and 350 rpm with an Eppendorf Thermomixer, the entire content was plated onto the LB agar plate containing 100 μg/ml ampicillin. The plates were then incubated at 37°C overnight. Next day, colonies from each constructs were picked for PCR confirmation of each construct using GoTaq DNA polymerase (Promega, Madison, WI, USA) and vector specific primers, and also for inoculation in the LB medium (with ampicillin) for overnight culture of each clone for mini-prep. The DNA mini-prep was performed using QIAprep Spin Miniprep Kit (QIAGEN, Valencia, CA, USA). All the cloned sequences were finally confirmed by automated DNA sequencing at the DNA lab of the Arizona State University using primers in the vectors.
Results and Discussion
As a proof of principle, we subcloned several cDNAs into different vectors (human nicotinic receptor (nAChR) α4, α9 and β2 subunits, and serotonin receptor type 3A (5-HT3A) subunit into the pGEMHE vector, human endothelial cell-specific molecule 2 (ECSM2) into the p3XFLAG-CMV-14 vector, and Akt3v1 or Akt3v2 into pLXSN vector). pGEMHE is a vector containing 5' and 3' untranslated regions (5'UTR and 3'UTR) of Xenopus β-globin, which is highly expressed in Xenopus oocytes. With this vector, the protein expression level of the inserted gene in Xenopus oocyte can be increased by up to 200-fold .
In addition, the PCR-based method could produce some unusual constructs. In our > 10-year practice with the QuickChange site-directed mutagenesis, we only encountered an unusual recombination once. With our FastCloning method in the past few months, we have not seen any unusual construct yet. Thus, if our cloning method produces unusual constructs, their occurrence must be very low. Figure 4D is an example of using restriction digestion to screen clones to exclude unusual constructs. It would be a good practice to perform such a screening for all resulting clones before DNA sequencing. With the optimized cloning conditions (Phusion DNA polymerase, 1:1 vector/insert ratio, 18 cycle PCR amplification, 2 μl of vector-insert mix for transformation, and 1 hour DpnI digestion), we have also successfully cloned other cDNAs into the target vectors, and obtained positive clones at efficiency ranging from 43% to 100%, with an overall efficiency > 70%. The results further validate the versatility and reliability of this new technique.
It is important to be aware that with PCR-based cloning, the synthesized primers may not be completely uniform with correct sequences. Random single-base deletions, mutations, or insertions can occur in a small fraction of primers, which result in unwanted deletion/mutation/insertion in a small fraction (< 5%) of clones. This is especially true for short insertion with long primers. Thus, sequencing across the primer region is required for ultimate confirmation of each clone. If random mutation happens in one clone, picking up another clone often solve the problem. The random mutation out of primer region is rare with high fidelity DNA polymerases. However, DNA sequencing of the entire coding region is still necessary. In the past 8 months, we have successfully used our new cloning method to obtain 21 constructs. Among the 63 sequenced clones, we found 2 deletions in 2 chimeras constructed with long primer pairs, but found no mutations in the entire regions of all constructs beyond primers. Finally, the cloning efficiency of our FastCloning method is high. Of the 21 cloning constructs, we obtained 19 desired constructs in single runs. In 2 experiments, we needed to repeat the procedure to get the final clones. With only one additional construct, we have not obtained final clones after two attempts.
We have developed a highly simplified and robust PCR cloning technique termed FastCloning. The new technique eliminates the need of PCR purification/gel purification kit and cloning kit. It is ligation-independent and does not require specific sequence in the vector. Thus, one can insert a DNA fragment into a vector at any desired position without considering restriction sites. This feature also makes it extremely easy to make constructs for fusion proteins and chimeras. In addition, it can be used to make short insertions and multiple mutations spanning a wide region (up to 120 bp) in a cDNA. Finally, it is a highly efficient and reproducible method.
List of abbreviations
polymerase chain reaction
nicotinic acetylcholine receptor
acetylcholine binding protein
gene name of the human nAChR α9 subunit
gene name of the human nAChR α4 subunit
gene name of the human nAChR β2 subunit
serotonin receptor type 3 A subunit
the gene name for human 5-HT3A receptor subunit
endothelial cell specific molecule 2 (also named ECSCR: endothelial cell-specific chemotaxis regulator)
acetylcholine binding protein
protein kinase B gamma variant 1 or 2
gene name of the human zinc activated cation channel
We thank Dr. Alan Gibson from the Barrow Neurological Institute for proofreading the manuscript. This work was supported by National Institute of Health (R01GM085237, to YC) and Barrow Neurological Foundation (to YC). This paper is subject to the NIH Public Access Policy.
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