Directional Topo Cloning

How Topoisomerase I Works

Topoisomerase I from Vaccinia virus binds to duplex DNA at specific sites and cleaves the phosphodiester backbone after 5´ -CCCTT in one strand (Shuman, 1991). The energy from the broken phosphodiester backbone is conserved by formation of a covalent bond between the 3´ phosphate of the cleaved strand and a tyrosyl residue (Tyr-274) of topoisomerase I. The phospho-tyrosyl bond between the DNA and enzyme can subsequently be attacked by the 5´ hydroxyl of the original cleaved strand, reversing the reaction and releasing topoisomerase (Shuman, 1994). TOPO Cloning exploits this reaction to efficiently clone PCR products (see diagram below).

The design of the PCR primers to amplify your gene of interest is critical for expression. Depending on the pET TOPO vector you are using, consider the following when designing your PCR primers:

• Sequences required to facilitate directional cloning (see below)
• Whether or not you wish to clone your PCR product in frame with the appropriate N-terminal and/or C-terminal peptide tag 

General Requirements for the Forward Primer

To enable directional cloning, the forward PCR primer must contain the sequence, CACC, at the 5´ end of the primer. The 4 nucleotides, CACC, base pair with the overhang sequence, GTGG, in each pET TOPO vector.

For example, below is the DNA sequence of the N-terminus of a theoretical protein and the proposed sequence for your forward PCR primer:
 
DNA sequence:    5´  -ATG GGA TCT GAT AAA
Proposed Forward PCR primer:    5´ -C ACC ATG GGA TCT GAT AAA

General Requirements for the Reverse Primer

In general, design the reverse PCR primer to allow you to clone your PCR product in frame with any C-terminal tag, if desired. To ensure that your PCR product clones directionally with high efficiency, the reverse PCR primer must not be complementary to the overhang sequence GTGG at the 5´ end. A one base pair mismatch can reduce the directional cloning efficiency from 90% to 75%, and may increase the chances of your ORF cloning in the opposite orientation. We have not observed evidence of PCR products cloning in the opposite orientation from a two base pair mismatch, but this has not been tested thoroughly.
 
Example: Below is the sequence of the C-terminus of a theoretical protein. You want to clone in frame with the C-terminal tag. The stop codon is underlined.

DNA sequence:    AAG TCG GAG CAC TCG ACG ACGGTG tag-3´

One solution is to design the reverse PCR primer to start with the codon just up-stream of the stop codon, but the last two codons contain GTGG (underlined below), which is identical to the overhang sequence. As a result, the reverse primer will be complementary to the overhang sequence, increasing the probability that the PCR product will clone in the opposite orientation. You want to avoid this situation.
 
DNA sequence:    AAG TCG GAG CAC TCG ACG ACG GTG tag-3´
Proposed Reverse PCR primer sequence:    TG AGC TGC TGC CAC-5´
 
Another solution is to design the reverse primer so that it hybridizes just down-stream of the stop codon, but still includes the C-terminus of the ORF. Note that you will need to replace the stop codon with a codon for an innocuous amino acid such as glycine or alanine.

• Remember that the pET TOPO vectors accept blunt-end PCR products. Refer below for a discussion of specific factors to consider when designing PCR primers for cloning into each pET TOPO vector.
• Do not add 5´ phosphates to your primers for PCR. This will prevent ligation into the pET TOPO vectors.
• We recommend gel-purifying your oligonucleotides, especially if they are long (> 30 nucleotides).
  

Example of Primer Design

The example below uses a theoretical protein and is for illustration purposes only. In this case, PCR primers are designed to allow cloning of the PCR product into pET101/D-TOPO. In this example, the N-terminus of the protein is encoded by:

 5´ -atggcccccccgaccgatgtcagcctgggggacgaa…

1. Design the forward PCR primer to be:
    5´ -caccatggcccccccgaccgat-3´

2. For the reverse primer, analyze the C-terminus of the protein.
       …GCG  GTT AAG TCG GAG CAC TCG ACG ACT GCA TAG-3´
       …CGC CAA TTC AGC CTC GTG AGC TGC TGA CGT   ATC-5´
3. To fuse the ORF in frame with the V5 epitope and 6xHis tag, remove the stop codon by starting with nucleotides homologous to the last codon (TGC) and continue upstream (underlined sequence in the bottom strand above). The reverse primer will be:
       5´ -TGC AGT CGT CGA GTG CTC CGA CTT-3´
4. This will amplify the C-terminus without the stop codon and allow you to clone the ORF in frame with the V5 epitope and 6xHis tag. If you don’t want the V5 epitope and 6xHis tag, simply begin with the stop codon:
       5´ -CTA TGC AGT CGT CGA GTG CTC CGA CTT-3´ 
    
   TOP

Introduction

Invitrogen Platinum SuperFi DNA Polymerase is a proofreading DNA polymerase that combines exceptional fidelity with trusted Platinum hot-start technology. Featuring >100x the fidelity of Taq polymerase, Platinum SuperFi DNA Polymerase is ideally suited for cloning, mutagenesis, and other applications that benefit from sequence accuracy.

Benefits of Platinum SuperFi DNA Polymerase include:

• Exceptional >100x Taq polymerase fidelity
• High specificity and increased yields with Platinum hot-start technology
• Robust amplification of difficult-to-amplify targets, including those of suboptimal purity or with ˃65% GC content
• Convenient workflow with room temperature reaction setup and 24-hour benchtop stability of pre-assembled reactions

Platinum SuperFi DNA Polymerase is engineered with a DNA-binding domain, resulting in high processivity and increased resistance to PCR inhibitors. This feature also enables fast-cycling protocols and amplification of long targets. The Platinum hot-start technology is based on proprietary antibodies that inhibit enzyme activity until the initial PCR denaturation step, preventing nonspecific amplification and primer degradation.

Platinum SuperFi DNA Polymerase is supplied with a separate vial of Invitrogen SuperFi GC Enhancer designed for GC-rich templates (>65% GC).

Protocol

The following procedure is suggested as a starting point when using Platinum SuperFi DNA Polymerase in  PCR amplification.

1. Program the thermal cycler as follows: 

¹ Important!Always use the T_m calculator on our website at thermofisher.com/tmcalculator to calculate the T_m of your primers and the recommended annealing temperature.

2. Add the following components to each PCR tube. (For multiple reactions, prepare a master mix of common components to minimize pipetting variation.)

   • Note: Consider the volumes for all components listed in steps 2 and 4 to determine the correct amount of water required to reach your final reaction volume.

   Component	50-μL rxn	Final conc.
   Water, nuclease-free	to 50 μL
   5X SuperFi Buffer1	10 μL	1X
   10 mM dNTP mix	1 μL	0.2 mM each
   5X Super GC Enhancer (optional) 2	10 μL	1X
   Platinum SuperFi DNA Polymerase	0.5 μL	0.02 U/μL

3. Mix and then briefly centrifuge the components.
4. Add your template DNA and primers to each tube for a final reaction volume of 50 µL.

¹ Optimal amount of low complexity DNA (plasmid, phage, BAC DNA) is 1 pg–10 ng per 50 μL reaction, but it can be varied from 0.1 pg to 50 ng per 50 μL reaction. Optimal amount of genomic DNA is 5–50 ng per 50 μL reaction, but it can be varied from 0.1 ng to 250 ng per 50 μL reaction.

5. Cap each tube, mix, and then briefly centrifuge the contents.
6. Place the tube in the thermal cycler and run the program from Step 1. After cycling, maintain the reaction at 4°C. Samples can be stored at –20°C until use.
7. Analyze products using Invitrogen E-Gel™ precast agarose gels or standard agarose gel electrophoresis. Visualize by staining with Invitrogen SYBR™ Safe DNA Gel Stain or ethidium bromide.

Introduction

Smearing, multiple banding, primer-dimer artifacts, or large PCR products (>3 kb) may necessitate gel purification. If you intend to purify your PCR product, be extremely careful to remove all sources of nuclease contamination. There are many protocols to isolate DNA fragments or remove oligonucleotides. Refer to Current Protocols in Molecular Biology, Unit 2.6 (Ausubel et al., 1994) for the most common protocols. Three simple protocols are provided below. Cloning efficiency may decrease with purification of the PCR product. You may wish to optimize your PCR to produce a single band.

Using the S.N.A.P.™ Gel Purification Kit

The S.N.A.P. Gel Purification Kit allows you to rapidly purify PCR products from regular agarose gels.

1. Electrophorese amplification reaction on a 1 to 5% regular TAE agarose gel.
2. Cut out the gel slice containing the PCR product and melt it at 65°C in 2 volumes of the 6 M sodium iodide solution.
3. Add 1.5 volumes Binding Buffer
4. Load solution (no more than 1 ml at a time) from Step 3 onto a S.N.A.P. column. Centrifuge 1 minute at 3000 x g in a microcentrifuge and discard the supernatant.
5. If you have solution remaining from Step 3, repeat Step 4.
6. Add 900 µl of the Final Wash Buffer.
7. Centrifuge 1 minute at full speed in a microcentrifuge and discard the flow-through.
8. Repeat Step 7.
9. Elute the purified PCR product in 40 µl of TE or sterile water. Use 4 µl for the TOPOCloning reaction.

Quick S.N.A.P. Method

An even easier method is to simply cut out the gel slice containing your PCR product, place it on top of the S.N.A.P. column bed, and centrifuge at full speed for 10 seconds. Use 1-2 µl of the flow-through in the TOPO™ Cloning reaction. Be sure to make the gel slice as small as possible for best results.

Low-Melt Agarose Method

If you prefer to use low-melt agarose, use the procedure below. Note that gel purification will result in dilution of your PCR product and a potential loss of cloning efficiency.

1. Electrophorese as much as possible of your PCR reaction on a low-melt agarose gel (0.8 to 1.2%) in TAE buffer.
2. Visualize the band of interest and excise the band.
3. Place the gel slice in a microcentrifuge tube and incubate the tube at 65°C until the gel slice melts.
4. Place the tube at 37°C to keep the agarose melted.
5. Add 4 µl of the melted agarose containing your PCR product to the TOPOCloning reaction.
6. Incubate the TOPOCloning reaction at 37°C for 5 to 10 minutes. This is to keep the agarose melted.
7. Transform 2 to 4 µl directly into One ShotTOP10 cells.

The cloning efficiency may decrease with purification of the PCR product. You may wish to optimize your PCR to produce a single band.

Introduction

Once you have produced the desired PCR product, you are ready to TOPOClone it into the pET TOPOvector and transform the recombinant vector into One Shot TOP10 E. coli. You should have everything you need set up and ready to use to ensure that you obtain the best possible results. We suggest that you read the this section and the next section entitled Transforming Chemically Competent Cells before beginning. If this is the first time you have TOPO Cloned, perform the control reactions (see Performing Control Reactions) in parallel with your samples.

Amount of PCR Product to Use in the TOPO Cloning Reaction

When performing directional TOPO Cloning, we have found that the molar ratio of PCR product:TOPO vector used in the reaction is critical to its success. To obtain the highest TOPO Cloning efficiency, use a 0.5:1 to 2:1 molar ratio of PCR product:TOPO vector. Note that the TOPO Cloning efficiency decreases significantly if the ratio of PCR product: TOP vector is <0.1:1 or >5:1 These results are generally obtained if too little PCR product is used (i.e. PCR product is too dilute) or if too much PCR product is used in the TOPO Cloning reaction. If you have quantitated the yield of your PCR product, you may need to adjust the concentration of your PCR product before proceeding to TOPO Cloning.

• Tip: For the pET TOPO vectors, using 1-5 ng of a 1 kb PCR product or 5-10 ng of a 2 kb PCR product in a TOPO Cloning reaction generally results in a suitable number of colonies.

Using Salt Solution in the TOPO Cloning Reaction

You will perform TOPO Cloning in a reaction buffer containing salt (i.e. using the stock salt solution provided in the kit). Note that the amount of salt added to the TOPO Cloning reaction varies depending on whether you plan to transform chemically competent cells (provided) or electrocompetent cells.

• If you are transforming chemically competent E. coli, use the stock Salt Solution as supplied and set up the TOPO Cloning reaction as directed below.
• If you are transforming electrocompetent E. coli, the amount of salt in the TOPO Cloning reaction must be reduced to 50 mM NaCl, 2.5 mM MgCl₂ to prevent arcing during electroporation. Dilute the stock Salt Solution 4-fold with water to prepare a 300 mM NaCl, 15 mM MgCl₂ Dilute Salt Solution. Use the Dilute Salt Solution to set up the TOPO Cloning reaction as directed below.

Performing the TOPO Cloning Reaction

Use the procedure below to perform the TOPO Cloning reaction. Set up the TOPO Cloning reaction depending on whether you plan to transform chemically competent E. coli or electrocompetent E. coli. Reminder: For optimal results, be sure to use a 0.5:1 to 2:1 molar ratio of PCR product:TOPO vector in your TOPO Cloning reaction.

• Note: The blue color of the TOPO vector solution is normal and is used to visualize the solution

*Store all reagents at -20°C when finished. Salt solution and water can be stored at room temperature or +4°C.

1. Mix reaction gently and incubate for 5 minutes at room temperature (22-23°C).
   • Note: For most applications, 5 minutes will yield plenty of colonies for analysis. Depending on your needs, the length of the TOPO Cloning reaction can be varied from 30 seconds to 30 minutes. For routine subcloning of PCR products, 30 seconds may be sufficient. For large PCR products (> 1 kb) or if you are TOPO Cloning a pool of PCR products, increasing the reaction time may yield more colonies.
2. Place the reaction on ice and proceed to Transforming Chemically Competent Cells.
   • Note: You may store the TOPO Cloning reaction at -20°C overnight.
     
   TOP

1. Innis, M. A., Gelfand, D. H., Sninsky, J. J., and White, T. S. (eds) (1990) PCR Protocols: A Guide to Methods and Applications, Academic Press, San Diego, CA