“Or how I stopped worrying and learned to love the sheep.” Biotechnology: “Or how I stopped worrying and learned to love the sheep.”

Restriction Enzymes Restriction enzymes are compounds first isolated in the 1970's They function by selectively cutting DNA at specific sequences

Restriction Enzymes These cuts usually occur in the following forms. The cut can be made straight across a base-pair sequence resulting in a "Blunt End“ The cut can be made in an offset manner leaving exposed nucleotide sequences. These exposed sequences are called "Sticky Ends" Blunt End Sticky end

Gene Splicing The presence of sticky ends allows segments of DNA to be joined together. Since DNA strands which have been cut by the same restriction enzyme can easily bond together according to base pairing rules.

Gene Splicing contd.. This allows for genes to be "cut & pasted" between organisms. This can be seen with production of human insulin. The DNA sequence of insulin is identified and cut out using a restriction enzyme. A plasmid from E. coli is removed and cut open using the same restriction enzyme Since both fragments have complimentary sticky ends the bind and the gene for human insulin is integrated into the plasmid The plasmid is then reinserted into a bacterial cell. This cell will produce insulin and is cultured. Human insulin can now be extracted and provided to diabetics.

Gel Electrophoresis Gel electrophoresis is a technique used to separate fragments of DNA. Separates fragments as a function of size. Most types use Agarose to separate fragments. Agarose is a porous gel. It can allow the passage of molecules through, however, larger molecules move more slowly through it since they cannot squeeze through the pores as easily as smaller molecules. Electrophoresis Apparatus

Electrophoresis Technique An agarose gel is casted with several holes called wells at one end. The gel is placed in an electrophoresis box which is filled with an electrolyte buffer solution. Samples of digested DNA are placed in the wells Electrical leads are attached to the ends of the box creating an electrical potential across the apparatus. Because DNA has a negative electrical charge. It is "pulled" towards the positive side of the apparatus. Also, since the smaller molecules travel faster through the agarose. Over time this separates the various sized fragments of DNA. The gel is then removed and stained for DNA. This results in a gel which shows several bands of stained DNA.

Finished Gel

Gel Electrophoresis

DNA Fingerprinting DNA is now a powerful tool in identification. Based on the fact that the amount of "junk DNA" differs uniquely between individuals. Structural genes are often separated by large regions of repeating basepairs. The number of these repeats is unique to an individual. Therefor when DNA from a person is cut with a restriction enzyme, the length of the fragments will be unique to an individual.

DNA Fingerprinting Contd… This will therefor produce a unique banding pattern following a gel electrophoresis. This test is highly accurate, and the probability of another individual possessing an identical banding pattern is estimated as around 1:14,000,000,000.

DNA Fingerprinting

Cloning

Cloning: What it is Cloning is the process of making a genetically identical organism through nonsexual means. It has been used for many years to produce plants (even growing a plant from a cutting is a type of cloning). Animal cloning has been the subject of scientific experiments for years, but garnered little attention until the birth of the first cloned mammal in 1997, a sheep named Dolly. Since Dolly, several scientists have cloned other animals, including cows and mice. The recent success in cloning animals has sparked fierce debates among scientists, politicians and the general public about the use and morality of cloning plants, animals and possibly humans Dolly, the first mammal clone

Dolly: A Mammal Clone Dolly In 1997, cloning was revolutionized when Ian Wilmut and his colleagues at the Roslin Institute in Edinburgh, Scotland, successfully cloned a sheep named Dolly. Dolly was the first cloned mammal. Wilmut and his colleagues transplanted a nucleus from a mammary gland cell of a Finn Dorsett sheep into the enucleated egg of a Scottish blackface ewe. The nucleus-egg combination was stimulated with electricity to fuse the two and to stimulate cell division. The new cell divided and was placed in the uterus of a blackface ewe to develop. Dolly was born months later.