BiotechnologyBiotechnology:““Or how I stopped worrying and Or how I stopped worrying and

learned to love the sheep.”learned to love the sheep.”

Restriction EnzymesRestriction Enzymes

• Restriction enzymes are compounds first Restriction enzymes are compounds first isolated in the 1970'sisolated in the 1970's

• They function by selectively cutting DNA at They function by selectively cutting DNA at specific sequences specific sequences

Restriction EnzymesRestriction Enzymes

• These cuts usually occur These cuts usually occur in the following forms.in the following forms.

• The cut can be made The cut can be made straight across a base-straight across a base-pair sequence resulting in pair sequence resulting in a "a "Blunt EndBlunt End““

• The cut can be made in The cut can be made in an offset manner leaving an offset manner leaving exposed nucleotide exposed nucleotide sequences. These sequences. These exposed sequences are exposed sequences are called "called "Sticky EndsSticky Ends""

Blunt EndBlunt End

Sticky endSticky end

Gene SplicingGene Splicing• The presence of The presence of

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

Gene Splicing contd..Gene Splicing contd..• This allows for genes to be "cut & This allows for genes to be "cut &

pasted" between organisms. This pasted" between organisms. This can be seen with production of can be seen with production of human insulin.human insulin.

• The DNA sequence of insulin is The DNA sequence of insulin is identified and cut out using a identified and cut out using a restriction enzyme.restriction enzyme.

• A plasmid from A plasmid from E. coliE. coli is removed is removed and cut open using the same and cut open using the same restriction enzymerestriction enzyme

• Since both fragments have Since both fragments have complimentary sticky ends the complimentary sticky ends the bind and the gene for human bind and the gene for human insulin is integrated into the insulin is integrated into the plasmidplasmid

• The plasmid is then reinserted into The plasmid is then reinserted into a bacterial cell. This cell will a bacterial cell. This cell will produce insulin and is cultured. produce insulin and is cultured. Human insulin can now be Human insulin can now be extracted and provided to extracted and provided to diabetics.diabetics.

Gel ElectrophoresisGel Electrophoresis• Gel electrophoresis Gel electrophoresis

is a technique used is a technique used to separate to separate fragments of DNA.fragments of DNA.

• Separates fragments Separates fragments as a function of size.as a function of size.

• Most types use Most types use AgaroseAgarose to separate to separate fragments.fragments.

• AgaroseAgarose is a porous is a porous gel. It can allow the gel. It can allow the passage of passage of molecules through, molecules through, however, larger however, larger molecules move molecules move more slowly through more slowly through it since they cannot it since they cannot squeeze through the squeeze through the pores as easily as pores as easily as smaller molecules.smaller molecules.

Electrophoresis ApparatusElectrophoresis Apparatus

Electrophoresis TechniqueElectrophoresis Technique • An agarose gel is casted with several An agarose gel is casted with several

holes called wells at one end.holes called wells at one end.• The gel is placed in an electrophoresis The gel is placed in an electrophoresis

box which is filled with an electrolyte box which is filled with an electrolyte buffer solution.buffer solution.

• Samples of digested DNA are placed Samples of digested DNA are placed in the wellsin the wells

• Electrical leads are attached to the Electrical leads are attached to the ends of the box creating an electrical ends of the box creating an electrical potential across the apparatus.potential across the apparatus.

• Because DNA has a negative Because DNA has a negative electrical charge. It is "pulled" towards electrical charge. It is "pulled" towards the positive side of the apparatus.the positive side of the apparatus.

• Also, since the smaller molecules Also, since the smaller molecules travel faster through the agarose. travel faster through the agarose. Over time this separates the various Over time this separates the various sized fragments of DNA.sized fragments of DNA.

• The gel is then removed and stained The gel is then removed and stained for DNA. This results in a gel which for DNA. This results in a gel which shows several bands of stained DNA.shows several bands of stained DNA.

Finished GelFinished Gel

Gel Electrophoresis

gel_electrophoresis.swf

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

DNA Fingerprinting Contd…DNA Fingerprinting Contd…

• This will therefor This will therefor produce a unique produce a unique banding pattern banding pattern following a gel following a gel electrophoresis.electrophoresis.

• This test is highly This test is highly accurate, and the accurate, and the probability of another probability of another individual possessing individual possessing an identical banding an identical banding pattern is estimated pattern is estimated as around as around 1:14,000,000,000. 1:14,000,000,000.

DNA FingerprintingDNA Fingerprinting

CloningCloning

Cloning: What it isCloning: What it is

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

Dolly, the first mammal cloneDolly, the first mammal clone

Dolly: A Mammal CloneDolly: A Mammal Clone

• DollyDolly

• In 1997, cloning was revolutionized In 1997, cloning was revolutionized when when Ian WilmutIan Wilmut and his colleagues and his colleagues at the Roslin Institute in Edinburgh, at the Roslin Institute in Edinburgh, Scotland, successfully cloned a sheep Scotland, successfully cloned a sheep named named DollyDolly. Dolly was the first . Dolly was the first cloned mammal. cloned mammal.

• Wilmut and his colleagues Wilmut and his colleagues transplanted a nucleus from a transplanted a nucleus from a mammary gland cell of a Finn Dorsett mammary gland cell of a Finn Dorsett sheep into the enucleated egg of a sheep into the enucleated egg of a Scottish blackface ewe. The nucleus-Scottish blackface ewe. The nucleus-egg combination was stimulated with egg combination was stimulated with electricityelectricity to fuse the two and to to fuse the two and to stimulate cell division. The new cell stimulate cell division. The new cell divided and was placed in the uterus of divided and was placed in the uterus of a blackface ewe to develop. Dolly was a blackface ewe to develop. Dolly was born months later. born months later.