Biotechnological Tools and Techniques.

Restriction Endonucleases (Restriction Enzymes)
- molecular scissors that can cut double-stranded DNA at a specific base-pair sequence
- each restricion endonucleases regonize a characteristic sequence of nucleotides known as 'recognition sites,' which are typically four to eight base pairs long
- palindromic because both strands have the same base sequence when read in the 5' to 3' direction
- ends of DNA fragments produced by a cut by different restriction endonucleases differ, depending on where the phosphodiester bonds are broken in the recognition sites
- the decrease in occurence of longer recognition sites results in fewer cuts - an important factor for molecular biolgoists who want to excise a piece of DNA that includes a whole gene.
- isolated and purified soely from bacteria
- provide a crude immune system
- around 200 of these restrictions endonucleases are avaliable commercially to molecular biologists

Gel Electrophoresis
- through taking advantage of the chemical and physical properties of DNA, gel electrophoresis seperates the unwanted fragments from the desired gene
- DNA fragments migrate through the gel at a rate that is inversely proportional to the logarithm of their size
- usually a square or rectangular slab and consists of a buffer containing electrolytes and agarose (gel-forming polysaccharide found in some types of seaweed that is used to form a gel meshwork for electrophoresis) or possibly polyacrylamide (aritifical polymer used to form a gel meshwork for electrophoresis)
- the DNA solutioon containing fragements to be seperated is mixed with a loading dye containing glycerol - a loading dye which allows visualization.
- using direct current, a negative charge is placed at one end of the gel where the wells are, and a positive charge is placed at the opposite end of the gel. The electrolyte solution conveys the charged electrode, with the shorter fragments migrating faster than the longer fragments, acheiving sepeartion
- small molecules can be visualized (they migrate ahead of all the DNA fragments) the electric current can be turned off before they reach the end of the gel.
- gel electrophoresis is not limited to the sepeartion of nucleic acids but is also commonly applied to proteins

Plasmids
- small, circular, double stranded DNA molecules lacking a protein coat that naturally exists in the cytoplasm of many starins of bacteria
- independent of the chromosome of the bacterial cell and range in size from 1000 to 200 000 base pairs
- carry genes that express proteins able to confer antibiotic resistance
- protect bacteria by carrying genes for resistance to toxic heavy metals, such as mercury, lead, or cadmium
- some bacteria carry plasmids possessing genes that enable the bacteria to break down herbicides, certain industeial chemicals or the components of petroleum
- relationship between bacteria and plasmids are endosymbiotic - both benefit from this mutual arrangement
- copy number: number of copies of a particular plasmid found in a bacterial cell
- engineered to contain a unique region that can be cut by many restriction enzymes
- after becoming recombinant DNA, a combination of the original plasmid DNA and the foreign DNA may now be introduced into a bacterial cell where it will be replicated to form many copies within the cell

Transformation
- the introduction of DNA from another source
- bacterium that has taken in a foreign plasmid is referred to as being transformed
- calcium ions neutralize the negative charge from the phosphate group on the plasmid DNA and on the phospholipids found in the cell membrane, minimizing the repelling effect of like charges, DNA can then enter the bacterial cell more easily
- ekectrioiratirs, chambers that subject the bacteria to an electric shock are also used to loosen the structure of the cell walls and allows foreign DNA to enter.