Restriction Digest and Ligation

Expression Plasmids History In the early 1970s, it was well known that some bacteria were resistant to certain antibiotics due to the presence of antibiotic resistance factors (genes) Researchers cloned the human insulin gene into a plasmid, transformed the plasmid into bacteria, and induced the bacteria to manufacture the human insulin protein Features of a Cloning Plasmid (1) Origin of Replication (ORI) The origin of replication is the location where DNA replication begins. This enables a plasmid to be reproduced, and therefore able to "survive" within the cells and to be passed on to daughter cells If a plasmid does not contain an ORI it will not be replicated, will not be passed on to daughter cells, and will eventually be lost over time in the population (2) Selectable marker gene Cloning plasmids usually have been engineered to contain a selectable marker which confers strong growth advantages to bacteria containing the plasmid versus those that do not The selectable markers are typically genes which confer resistance (i.e. The ability to grow in the presence of) to antibiotics such as kanamycin, ampicillin, carbenicillin, and tetracyclines (3) Multiple cloning site (MCS), polylinker, or polycloning site Multiple cloning site (MCS), polylinker, or polycloning site are all terms describing the same thing: a location on a plasmid where there are several unique restriction enzyme cut sites Polylinkers have been engineered by scientists into cloning plasmids to make insertion of target DHA easier and more efficient (4) Regulators of transcription and translation In order to express a gene of interest, a plasmid must contain the regulatory sites for transcription and translation If these sites do not exist or if the gene of interest is inserted incorrectly into the plasmid the RNA molecule or the protein may not be produced Restriction Enzymes Introduction Most prokaryotic organisms have restriction modification systems that prevent recombination with foreign DNA and allow recognition of DNA from an identical species A modification enzyme (such as a DNA methyltransferase or DNA methylase) chemically modifies a specific residue in a particular sequence A restriction enzyme, on the other hand, recognizes specific DNA sequences and acts as an endonuclease to cleave the DNA (i.e. by breaking a phosphodiester bond) Restriction Modification System When the sequence is modified by the modification enzyme, the restriction enzyme can not cleave DNA because the modification prevents it from binding Bacterial cells use the restriction modification system to protect their DNA by modification and cleave foreign DNA using restriction enzymes, reducing the chances of recombination with other bacterial strains and the chances of infection by viral DNA Restriction Enzymes By cleaving the phosphodiester bond of HindIII, the enzyme creates what is known as a sticky end. This means each end of the DNA fragments formed by the cleavage reaction Will have overhangs (one will have an overhang on the bottom strand while the other will have the overhang on the top strand) While many restriction enzymes cleave DNA in a manner similar to HindIII, and form sticky ends, others cleave DNA at the same location on both strands thereby creating "blunt ends" Compatible Cohesive Ends Generally, unless further modification is done, only pieces of DNA that have been cleaved with "compatible" restriction enzymes can be joined or ligated together On the other hand, DNA cut by "blunt-end" enzymes can be lighted together regardless of which bluntend enzyme was used. Because of this, sticker end restriction enzymes are often used in order to help limit which pieces of DNA will be cloned together Enzymatic Reaction of Restriction Enzymes There are three categories of restriction enzymes: type I, which recognizes specific DNA sequences but then make seemingly random cuts sometimes as far as 1000 base pairs away from the recognition site; type II, which recognize and cut directly in the recognition sequence; and type III, which recognize specific sequences but make cuts about 25 bases away from the recognition sequence Type II enzymes bind to the DNA, scan the DNA, and when they recognize a specific DNA sequence, they cut both strands of the DNA molecule Type II restriction enzymes do not require ATP hydrolysis. Instead, the binding of the enzyme at the recognition sequence induces a conformational change, both in the enzyme and the DNA molecule