|Cloning using restriction enzymes|
|Restriction enzymes (restriction endonucleases) are proteins that cut DNA at (or close to) specific recognition sites (see the catalogs of manufacturers or the Restriction Enzyme Database). Two types of restriction enzymes exist that differ in the way they cut the target DNA:
Blunt end cutters. These enzymes cut both strand of the target DNA at the same spot creating blunt ends.
Sticky end cutters. These enzymes cut both strand of the target DNA at different spots creating 3'- or 5'-overhangs of 1 to 4 nucleotides (so-called sticky ends).
To be able to clone a DNA insert into a cloning or expression vector, both have to be treated with two restriction enzymes that create compatible ends. At least one of the enzymes used should be a sticky end cutter to ensure that the insert is incorporated in the right orientation.
For our cloning work, we normally use two sticky end cutters that create different 5'-overhangs:
3'-end: we use Nco I because its recognition site contains the ATG start codon.
|Methylation of DNA|
Digestion of vector DNA using (preferably) two restriction enzymes.
Dephosphorylation of the ends using calf intestine or shrimp alkaline phosphatase. This reduces the background of non-recombinants due to self-ligation of the vector (especially when a single site was used for cloning).
Purification of the digested vector by agarose electrophoresis to remove residual nicked and supercoiled vector DNA and the small piece of DNA that was cut out by the digestions. This usually reduces strongly the background of non-recombinants due to the very efficient transformation of undigested vector.
Digestion of insert DNA using (preferably) two restriction enzymes.
Purification of the digested insert. Purification should be carried out by agarose gel electrophoresis when the insert is subcloned into a vector from a vector with the same selective marker or PCR amplified from a vector with the same selective marker. Otherwise, it can be purified using a commercial kit (such as Qiagen's PCR purification kit).
The next step is the ligation of the insert into the linearized vector. This involves the formation of phosphodiester bonds between adjacent 5'-phosphate and 3'-hydroxyl residues, which can be catalyzed by two different ligases: E. coli DNA ligase and bacteriophage T4 DNA ligase. The latter is the preferred enzyme because it can also join blunt-ended DNA fragments.
The efficiency of the ligation reaction depends on:
For pET vectors, good results are obtained at a vector DNA concentration or approx. 1 nM (i.e. 50-100 ng vector DNA per 20-ml ligation mix).
The ratio between vector and insert DNA. The maximum yield of the right recombinants is usually obtained using a molar ratio of insert to vector DNA of approx. 2. If the concentration of insert DNA is substantially lower than that of the vector, the ligation efficiency becomes very low.
The cloning strategy. Higher yields of the right recombinant are obtained when the vector and insert have been prepared using two restriction enzymes and the digested vector has been gel-purified before the ligation reaction (as shown in the figure on the right).
high concentrations of blunt-ended DNA fragments.
Sambrook, J., Fritsch, E.F. & Maniatis, T. (1989) Molecular Cloning: A laboratory manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp 1.63-1.70.
pET System Manual (1999), 8th Ed., Novagen.