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.
It will save you a lot of time when you could carry out the two digestions simultaneously (double digestion). Not all restriction enzymes work equally well in all commercially available buffers and, therefore, it is worthwhile to check (e.g. in the reference appendix of the New England Biolabs catalog) which enzymes are compatible and in which buffer. To ensure efficient digestion the two recognition sites should be more than 10 base pairs apart. If one of the enzymes is a poor cutter or if the sites are separated 10 base pairs or less, the digestions should be performed sequentially. The first digest should be done with the enzyme that is the poorest cutter and the second enzyme added after digestion has been verified by running a sample of the reaction mix on an agarose gel.
For our cloning work, we have selected two sticky end cutters that create different 5'-overhangs:
- 3'-end: Nco I. Its recognition site contains the ATG start codon.
- 5'-end: BamH I. It is cheap and active in most buffers.
It is recommended to use the special BamH I buffer (New England Biolabs) for this double digestion.
- 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:
- the absolute DNA concentration. The concentration should be high enough to ensure that intermolecular ligation is favored over self-ligation but not so high as to cause extensive formation of oligomeric molecules.
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.
In practise, we set-up ligation reactions with a molar ratio of insert to vector DNA of 1:1, 2:1, and 3:1.
- 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).
The ligation of blunt-ended fragments is less effective than that of sticky-ended ones. Blunt-end ligation may be enhanced by:
- high concentrations of blunt-ended DNA fragments.
- a high concentration of ligase (10,000 NEB units/ml).
- a low concentration of ATP (0.1 mM).
- the addition of PEG 4000 [5% (w/v)].
Reference: Pheiffer, B.H. & Zimmerman, S.B. (1983) Nucleic Acid Res. 11, 7853-7871.
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