Some thermostable DNA polymerases, including Taq, add a single nucleotide base extension to the 3′ end of amplified DNA fragments. These polymerases usually add an adenine, leaving an “A” overhang. There are several approaches to overcome the cloning difficulties presented by the presence of A overhangs on PCR products. One method involves treating the product with Klenow to create a blunt-ended fragment for subcloning. Another choice is to add restriction sites to the ends of your PCR fragments. You can do this by incorporating the desired restriction sites into the PCR primers. After amplification, the PCR product is digested and subcloned into the cloning vector. Take care when using this method, as not all restriction enzymes efficiently cleave at the ends of DNA fragments, and you may not be able to use every restriction enzyme you desire. There is some useful information about cutting with restriction sites close to the end of linear fragments in the Restriction Enzyme Resource Guide. Also, some restriction enzymes require extra bases outside the recognition site, adding further expense to the PCR primers as well as risk of priming to unrelated sequences in the genome.
The easiest method, and the method of choice for cloning PCR products, is T-vector cloning. This method takes advantage of the “A” overhangs on the PCR product. T vectors are linearized plasmids that have been treated to add 3′ T overhangs to match the A overhangs of the amplicon. The PCR fragment is directly ligated to the T-tailed plasmid vector with no need for further enzymatic treatment other than the action of T4 DNA ligase. If desired, the insert can then be easily transferred from the T vector to other plasmids using the restriction sites present in the T vector.
Proofreading polymerases like Pfu and Tli do not add “A” overhangs, and PCR products generated with these polymerases are blunt-ended. Here is a simple method for adding an A-tail to DNA fragments generated by these polymerases to enable T-vector cloning.
A-Tailing Reaction for Blunt-Ended Products
An A residue can is added by incubating the PCR fragment with dATP and a nonproofreading DNA polymerase, which will add a single 3′ A residue. Blunt ended restriction digest fragments can also be A-tailed using this method. The method below uses GoTaq Flexi DNA Polymerase (comes with a Mg-free reaction buffer), but any Taq DNA polymerase can be used.
Set up the following reaction in a thin-walled PCR tube:
1–4µl purified blunt-ended DNA fragment (from PCR or restriction enzyme digestion)
2µl of 5X GoTaq Flexi Reaction Buffer (Colorless or Green)
2µl of 1mM dATP (0.2mM final concentration)
2µl GoTaq Flexi DNA Polymerase (5u/µl)
0.6µl of 25mM MgCl2 (1.5mM final concentration)
Nuclease-free water to a final volume of 10µl
Incubate at 70°C for 15–30 minutes in a water bath or thermal cycler. After the tailing reaction is finished, 1–2µl can be used without further cleanup for ligation into the pGEM-T or pGEM-T Easy Vector Systems.
Tips for T-Vector Cloning Experiments
Although T-vector cloning experiments are simple, there are a few things to be careful of to ensure best results:
- Avoid introduction of nucleases, which may degrade the T overhangs on the vector. Use sterile, nuclease-free water in your ligation reactions.
- Use high-efficiency competent cells (≥1 × 108cfu/μg DNA) for transformations. The ligation of fragments with a single-base overhang can be inefficient, so it is essential to use cells with a transformation efficiency of at least 1 × 108cfu/μg DNA to obtain a reasonable number of colonies.
- Limit exposure of your PCR product to shortwave UV light to avoid formation of pyrimidine dimers. Use a glass plate between the gel and UV source. If possible, only visualize the PCR product with a long-wave UV source.
More details on PCR cloning are available in the following resources:
- T Vectors Technical Manual
- Properties of Thermostable Polymerases
- Protocols & Applications Guide, Cloning Chapter
Analysis of molecular biology experiments such as cloning and PCR often involve agarose gel electrophoresis. Here are two videos that show you how to get the most from your gels.