May 20 1990
Matthew S. Holt
Under natural conditions many plasmids are transmitted to a new host by a process known as bacterial conjugation. Newer plasmid vectors however lack the nic/bom site and cannot be conjugated. In the laboratory plasmid DNA can be introduced into modified bacteria (called competent cells) by the process of transformation. Even under the best circumstances plasmids become stably established in a small minority of the bacterial population. Transformants can be easily identified by the selectable marker encoded by the plasmid i.e. antibiotic resistance and the resulting phenotype of those bacterial cells.
Uncut plasmid DNA can be in any of five forms - nicked circular linear covalently closed supercoiled or circular single-stranded. When run on a gel one frequently will see these forms as different bands. The exact distances between the bands of these different forms is influenced by percentage of agarose in the gel time of electrophoresis degree of supercoiling and the size of the DNA. One cannot accurately determine size from uncut plasmid DNA. When cut with an enzyme with one recognition site in the plasmid almost all the DNA will fall in one band which equals the linear size of the plasmid (see illustration below).
Almost all research plasmid vectors contain a closely arranged series of synthetic restriction sites called the "polylinker". In most cases these restriction sites are unique to the polylinker and hence provide a variety of targets that can be used singly or in combination to clone foreign DNA fragments. The most recent phase of construction of plasmid vectors involves the incorporation of ancillary sequences that are used for a variety of purposes including generating single-stranded DNA templates for DNA sequencing transcription of foreign DNA sequences in vitro and expression of large amounts of foreign protein in vitro.
Fragments of foreign DNA can be cloned in a linearized plasmid vector bearing compatible ends by the activity of Bacteriophage T4 DNA Ligase. The enzyme will catalyze the formation of a phosphodiester bond between adjacent nucleotides if one nucleotide contains a 5'-phophate group and the other nucleotide contains a 3'-hydroxyl group.
During the ligation reaction however the vector can recircularize without the insert DNA. This can be minimized by removing the 5'-phosphate from both termini of the linear vector with Calf Intestinal Alkaline Phosphatase (CIP). Described in the following procedures is a method that has worked consistently. It is recommended to run the vector on a gel cut out the vector band and gel elute to separate the vector from any uncut DNA.The smallest amount of uncut vector will transform efficiently and make finding the bacteria containing the recombinant plasmid virtually impossible. CIP has no effect on uncut circularized plasmids.
A fragment of foreign DNA can be inserted into the vector by a process knows as directional cloning by cutting the vector polylinker with two unique restriction sites. Because of the lack of complementary ends the vector fragment cannot circularize efficiently. The insert DNA however must also have the same cohesive termini as the vector. Directional cloning is widely used when insert DNA orientation is specific. Directional cloning also depletes the number of available cloning sites for further constructing. Specific orientations can be obtained by screening several recombinants from a single-enzyme ligation until the desired orientation is identified.
Another method of ligation though substantially less efficient is blunt-end ligations. Some restriction enzymes cleave both strands of DNA resulting in blunt ends - i.e. no cohesive overhang. Both the insert and vector termini resulting from any restriction enzyme digest can be made blunt. One method utilizes T4 DNA Polymerase and dNTPs to fill in the overhang. Another method uses the enzyme Mung Bean Nuclease to cleave the single stranded overhang. Ligation reactions involving blunt-ended molecules require much higher concentrations of both vector and insert DNA and T4 DNA ligase. One end blunt ligations are slightly more efficient (and directional) and can be achieved by first blunting the linear DNA and then digesting with a restriction enzyme unique to only one end of the DNA. Again both vector and insert must have complementary ends. Either of these methods may change the DNA sequence resulting in the loss of the restriction site.
When it is impossible to find a suitable match between restrictions sites in the plasmid and those at the ends of the insert synthetic linkers can be ligated to the linearized DNA. The procedure requires blunt-end linear DNA fragments to which ligase adds a series of these
synthetic restriction sites. By the digesting with that specific restriction enzyme the resulting DNA has only one linker on each end because the DNA-linker bond is not a restriction site.
Certain restriction overhangs can be modified using Klenow and the recessed 3' termini can be partially filled to generate complementary restriction overhands that are otherwise incompatible. For instance an insert with BamH1 recognition site G'GATC C C CTAG'G has the overhang GATC after digesting. By filling in with dCTP and dTTP and Klenow the resulting overhand is AG. This is compatible with an Acc1 overhang of AG. Note however that the sequence resulting from this ligation is neither a BamH1 site nor an Acc1 site and an alternative method must be used to cut out the insert.
Plasmid Vector Preparation
10 mM ZnCl2 10 mM MgCl2 100 mM Tris pH 8.0
0.5 M Tris pH 7.4 0.1 M MgCl2 0.1 M DTT 10 mM ATP 10 mM Spermidine
Sambrook J. Fritsch E.F. and T. Maniatis.(1989) Molecular Cloning A Laboratory Manual. Second edition. Cold Spring Harbor Laboratory Press pp.1.53-1.69.