Li C et al. (2011), FastCloning: a highly simplified, purification-...

XB-ART-44827

BMC Biotechnol 2011 Oct 12;11:92. doi: 10.1186/1472-6750-11-92.

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FastCloning: a highly simplified, purification-free, sequence- and ligation-independent PCR cloning method.

Li C , Wen A , Shen B , Lu J , Huang Y , Chang Y .

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BACKGROUND: Although a variety of methods and expensive kits are available, molecular cloning can be a time-consuming and frustrating process. RESULTS: 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. CONCLUSION: 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.

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Species referenced: Xenopus laevis
Genes referenced: chrna4 kit

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	Figure 1. The procedures for FastCloning: Step 1. PCR amplification of vector and insert. Note that the primer pair for insert amplification has 16-base tails overlapping with the PCR-amplified vector ends. Step 2. DpnI digestion. The parent DNA templates (if in a plasmid) for PCR amplification needs to be methylated in order to be compatible to DpnI digestion. Although the detailed mechanism is not known, it is likely that the 3' exonuclease activity of the high fidelity DNA polymerase directly creates sticky ends for the overlapped regions of the vector and insert during DpnI digestion, allowing them to form a circular construct with nicks. Step 3. transformation into competent E. coli. cells. The nicks will be repaired after transformation into the bacteria.
	Figure 2. Chimera construction or insertion. Top left: PCR amplification of both ends of the parent cDNA along with the vector. For chimera construction, one fragment of the cDNA needs to be replaced. Thus, the forward primer is immediately downstream of the fragment to be substituted, and the reverse primer will be immediately upstream of the fragment to be substituted. For insertion, however, two primers will be next to each other without skipping a single base. Top right: Insert amplification of the equivalent region of a homologous gene (cDNA) for chimera construction. However, for insertion of a cDNA encoding a full length protein, such as green fluorescent protein, the insert amplification will cover the entire cDNA. The remaining procedure is the same as in Figure 1.
	Figure 3. Chimera construction or insertion with a short DNA fragment. To replace a short stretch of DNA, such as a DNA fragment encoding a transmembrane region of a nAChR subunit for chimera construction, or to insert a short tag, such as FLAG-tag, into some part of a protein, amplification of the insert is not necessary. In this case, the insert can be directly included in two primers for single PCR amplification of the cDNA along with the vector. The forward primer starts immediately downstream of the insertion site and has a tail with the 3' part of the insert. The reverse primer starts immediately upstream of the insertion site and has a tail with 5' part of the insert. Two primers only require ~16-base overlap. Thus, for a 120 bp insertion, each primer needs to have a 68-base tail for insertion and ~17-22 bases for annealing (depending on the GC content). The total length of each primer will be about 85-90 bases. DpnI digestion and transformation are the same as in Figure 1.
	Figure 4. Optimization of cloning conditions. (A) PCR amplification of a target cDNA (human nAChR α9 subunit) and the pGEMHE vector using different DNA polymerases: Pfu Turbo, PfuUltra and Phusion. (B) Comparison of number of colonies grown on the plates after transformation. Three different vector-to-insert ratios (1:1, 1:2, and 1:4) during DpnI digestion and three amounts of vector-insert mixtures (2, 4, and 8 μl) for transformation were tested. See text for details. (C) Clone validation by PCR using GoTaq DNA polymerase. Lanes 1 to 12: target clones to be validated; Lane 13: 1 Kb plus DNA ladder; Lane 14: pGEMHE vector control; Lane 15: negative control using pCR4-TOPO-α9 parent plasmid. (D) Clone validation by restriction digestion to exclude unusual constructs. Lane 1: 1 Kb plus DNA ladder, Lanes 2-11: target clones double digested with KpnI and NheI. Note that this digestion resulted in a pGEM vector and an insert with α9 nAChR plus the 5'UTR and 3'UTR of Xenopus β-globin.
	Figure 5. An example of chimera construction with a short (105 bp) DNA fragment replaced by a synthesized insert of 99 bp (included in two primers). (A) A region of α7 nAChR subunit sequence with a 105 bp fragment (blue) to be replaced. (B) Amino acid sequence alignment of human α7 nAChR and corresponding region of Aplysia californica AChBP. Segments to be replaced are colored with blue (35 codons) for α7 nAChR sequence and red (33 codons) for AChBP sequence. (C) Target chimera construct with the human nAChR α7 subunit sequence (black) and a substituted 99 bp DNA fragment (red) from the mammalian codon-optimized homologous sequence of the AChBP. Two colored arrows indicate two primers with a 15 bp overlapping region (highlighted). Note that the entire insert of the 99 bp fragment is included in the two primers. Thus, there is no need to amplify the insert. The length of each primer is 81 bases for the forward primer and 76 bases for the reverse primer.
	Figure 6. An example of making multiple mutations across a wide region. (A) A fragment of the human nAChR α2 subunit cDNA with 8 arginine (R, in bold) codons (top) to be substituted by glutamine (Q) codons (bottom). Mutated nucleotides are indicated in red. (B) Actual forward (45-base) and reverse (46-base) primers with 16 bp overlapping in their 5' ends.

References [+] :

Aslanidis, Ligation-independent cloning of PCR products (LIC-PCR). 1990, Pubmed