Restriction Endonucleases
- Restriction endonucleases (aka restriction enzymes) - enzymes that are able to cleave double-stranded DNA into fragments at specific sequences, known as recognition sites
- Recognition site - a specific sequence within double-stranded DNA, usually palindromic and consisting of 4 to 8 nucleotides, that a restriction enzyme recognizes and cleaves
- Restriction endonucleases produce DNA fragments with both sticky ends and blunt ends.
- Sticky ends - fragment ends of a DNA molecule with short single-stranded overhangs, resulting from cleavage by a restriction enzyme. These are more useful to molecular biologists, since they can be joined more easily to other sticky-end fragments produced by the same restriction endonuclease.
- Blunt ends - fragment end of a DNA molecule that are fully base paired
- Frequency of cuts of a restriction endonuclease depends on the length of their recognition sites. The more base pairs there are in the recognition site, the lower the frequency of cuts.
- Restriction enzymes are produced from bacteria, who use them to defend against the foreign DNA of viruses.
Gel Electrophoresis
- Gel electrophoresis - separation of charged molecules on the basis of size by sorting through a gel meshwork
- Each nucleotide has the same charge-to-mass ratio. The only difference between fragments of DNA of differing lengths is the number of nucleotides.
- Gel electrophoresis is like a molecular sieve. A shorter fragment will travel through the gel faster because it is able to navigate through the pores of the gel more easily. Longer fragments travel more slowly through the gel because they have a harder time moving through the pores of the gel.
- Solution containing DNA fragments are placed in a well in the gel. Gel consists of a buffer containing electrolytes and agarose, or polyacrylamide. Using direct current, a negative charge is placed at the end of the gel where the wells are, and a positive charge is placed at the opposite end of the gel. DNA will migrate down towards the positively charged electrode, with the shorter fragments migrating faster than the longer ones.
- After process is complete, gel is stained. Most commonly used stain is ethidium bromide, a molecule that fluoresces under UV light. Size of fragments can be determined using a molecular marker as a standard. Desired fragments can also be excised out of the gel for further study.
- Also applied to proteins, using polyacrylamide gels because they have smaller pores and proteins are generally smaller in size than nucleic acids.
Plasmids
- Plasmids are small circular pieces of DNA that can exit and enter bacterial cells. Using bacterial enzymes and ribosomes, DNA contained in plasmids can be replicated and expressed.
- Bacteria benefit from presence of plasmids. Plasmids carry genes for antibiotic resistance, resistance to toxic heavy metals, and the ability to break down certain chemicals. Relationship between bacteria and plasmids is endosymbiotic.
- Plasmids have a copy number - the number of copies of a particular plasmid found in a bacterial cell. More copies of a plasmid lead to more protein being synthesized.
- Artificial plasmids may be engineered to contain a multiple-cloning site - a region in the plasmid that has been engineered to contain recognition sites of a number of restriction endonucleases. Recognition sites are present only once, so only one cut can be made in the DNA.
- If foreign gene was excised using the same restriction enzyme, it will have the same complementary ends as the cut plasmid. When placed together, the sticky fragments will anneal. The foreign gene will permanently become part of the plasmid after the phosphodiester bonds are re-established with DNA ligase. Plasmid is now recombinant DNA. It can be introduced into bacterial cells, where it replicates to form many copies, thereby cloning the gene.
Transformation
- Transformation - introduction of foreign DNA, usually by a plasmid or virus, into a bacterial cell
- Plasmids can be used as vectors - vehicles by which DNA may be introduced into host cells
- Competent cell - a cell that readily takes up foreign DNA. Most bacteria are not naturally competent, but can be chemically induced to become so by being treated with a solution of calcium chloride at 0 degC, adding the plasmids, and then subjecting the solution to a quick heat shock treatment at 42 degC for 90 seconds, creating a draft that sweeps the plasmids into the bacterial cells.
- Selective plating is a method used to isolate cells with recombinant DNA. The plasmid vector also contains an antibiotic-resistance gene. The successfully transformed bacteria will be able to grow on media that contains the antibiotic. To check if the gene exists in the transformed bacteria, colonies are grown until enough plasmid DNA can be extracted. Plasmid DNA is subjected to restriction enzyme digestion to release cloned DNA fragment. DNA is put through gel electrophoresis. If expected pattern of bands appears on the gel, then the colony carries the recombinant DNA plasmid with the desired gene.
- New methods of transformation include electroporators and electrical "gene guns".
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