Important enzymes in cloning and molecular biology:
DNA polymerase [local]- elongates DNA from the 3' end by adding dNTP (deoxynucleoside biphosphate) units to free 3'-OH. Requires a template strand of DNA, a 'primer' with a free 3'-OH and the 4 dNTPs as substrates. There are actually several of these enzymes (e.g. Pol I, Klenow fragment, Taq) and they have different functions. These enzymes are used for DNA replication and repair of DNA.
RNA polymerase [local]--elongates RNA from the 3'-end by adding NTP units to free 3'-OH. Does not require a primer, but does utilize DNA template and the 4 NTPs as substrates. This is the enzyme used for cellular transcription. Can be used to prepare labeled RNA probes for hybridization.
RNA-dependent DNA polymerase (reverse transcriptase [local])-- makes a DNA strand from an RNA template. Originally found in retroviruses. Used in the first step of making cDNA.
Nucleases-- enzymes that hydrolyze polynucleotides. Usually, but not always, leave a 5'-P and 3'-OH.
Endonuclease-- an enzyme that hydrolyzes a polynucleotide in the middle of the molecule
Exonucleases- an enzyme that hydrolyzes the terminal residue from a polynucleotide.
DNase-- an enzyme that hydrolyzes DNA; usually not specific for a base sequence.
Restriction endonuclease-- class of bacterial enzymes that cleave both strands of a DNA molecule within a specific recognition sequence. May leave either blunt or 'sticky' ends. An essential component of cloning procedures.
RNase-- an enzyme that hydrolyzes RNA. Used during cDNA cloning to convert the RNA-DNA duplex to the ssDNA. Also used to degrade RNA present in preparations of bacterial plasmids.
DNA ligase [local]-- an enzyme that takes a DNA with an open phosphate-sugar gap and creates a covalent closure.
Phosphatase-- an enzyme that removes the 5'-P from a polynucleotide. Used to treat linearized plasmids so that they can't religate without accepting an insert of cloned DNA.
The piece of DNA that is used to carry the piece of foreign DNA is called a vector [local]. Generally, it has a restriction enzyme site [local] into which the foreign DNA is inserted. There are a variety of cloning vectors [local].
Plasmids are circular pieces of DNA that contain origins of replication and can replicate in bacteria independent of the chromosomal DNA. Plasmids often contain genes which confer resistance to antibiotics like ampicillin and tetracycline.
1970s vectors used disruption of an antibiotic resistance gene to identify the presence of inserted foreign DNA.
Example: pBR322; restriction map [local]. The problem with these plasmids was that they had relatively few restriction endonuclease sites. This was a severe limitation because you can't clone in an insert if it has an internal restriction site for one of these enzymes.
Plasmids developed more recently are often engineered to contain a region with polylinker regions containing unique sites for a multiple restriction endonculeases. These plasmids also contain an antibiotic resistance gene to permit selection of bacteria containing plasmids, as well a marker genes to demonstrate if the plasmid has an inserted piece of foreign DNA cloned into it. An example of such a plasmid is pUC19 Cartoon [local]; restriction map [local]).
This type of plasmid also makes use of an enzyme (beta-galactosidase) which acts as a reporter. If the bacteria is Ampr, but has beta-galactosidase activity, then it contains no insert. If it has lost beta-galactosidase activity, then the gene has been disrupted by the insertion of foreign DNA. This is the basis for Blue/White screening. At the start of the beta-galactosidase gene is the lac promoter. In lactose metabolism, it is normally induced by allo-lactose, but in the laboratory we induce it with a nonhydrolyzable analog, isopropylthio-beta-D-galactopyranoside (IPTG [local]). Also, rather than using lactose as the substrate for the beta-galactosidase, an chromogenic analog called X-gal is used. When hydrolyzed it forms an insoluble, blue indole derivative that marks the colonies containing the beta-galactosidase activity. After transformation of E. coli, the cells are plated on an ampicillin-containing medium overlaid with IPTG and X-gal. The cells that grow all contain the plasmid. The blue colonies have no insert. The white colonies contain a foreign DNA insert and can be picked from the plate for further study.
More recently, plasmids have been developed as shuttle vectors to move DNA between different organisms. These need to contain origins of replication for both types of organisms.
Also useful are expression vectors. These have a multiple cloning site downstream from a strongly inducible promoter like lac or ptac. When induced, the protein is often expressed at levels of 10 to 40% of the total E. coli protein. These usually come as sets of three with a one base length difference between the promoter and the start of transcription so that it is possible to get your protein coding sequence 'in frame' with the promoter. It is also common to use expression vectors that make fusion proteins. This is where your protein of interest is fused on the N- or C-terminal end to another protein. This is done for two reasons. First, the extra fusion protein is a tag that can be used in the purification of your protein. The most common is a poly-His sequence fused to your protein of interest. Poly-his is a polymer of the amino acid histidine. This poly-His binds to chromatography columns with adsorbed nickel ions and is selectively purified in one step. Another is the maltose-binding protein which has a high affinity for alpha-galactoside polymers. A second reason for making a fusion protein is that very often foreign proteins don't fold properly in E. coli and are insoluble. They tend to pellet with the cell debris in 'inclusion bodies [local]' and are hard to recover. By fusing them to a soluble E. coli protein like the maltose-binding protein [local] , they are much more soluble.
Other cloning vectors include cosmids [local] for larger pieces of DNA than is usually possible with plasmids, bacteriophage lambda [local], shuttle vectors to move recombinant DNA between two different organisms, and artificial chromosomes.
What else can you do with cloned DNA?
