Restriction enzymes

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10 Δεκ 2012 (πριν από 4 χρόνια και 8 μήνες)

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Genetic Engineering

• All DNA is the same in all organisms in terms of basic structure
• Because of this DNA can be transferred between species
• When DNA from one species is inserted into another, this is called recombinant DNA
• Restriction enzymes are obtained from bacteria and they cut DNA at specific sites
• Bacteria use them to cut up viral DNA and thus neutralize the virus
• Some of these enzymes cut DNA in a staggered fashion
• For example, the restriction enzyme EcoR1 cuts as follows:
G A A T T C
C T T A A G

• This leaves “sticky ends”
G A A T T C
C T T A A G
• The sticky ends can form bonds to place the strand together again if they come in
contact
• Once back together DNA ligase can form covalent bonds to seal them back together
• Bacteria have small rings of DNA known as plasmids
• Human (and other) genes can be spliced into plasmids using restriction enzymes and
DNA ligase

Steps in Cloning a Gene
• Cut plasmid with a restriction enzyme
• Cut DNA containing the gene to be cloned with the same restriction enzyme
• Both now have complementary sticky ends
• Mix DNA and plasmids
• The DNA couples to the plasmids
• Place plasmid into bacteria
• As bacteria divide the gene will be copied and passed along-all identical copies
(clones)
• After millions of copies are made the same restriction enzyme can be used to cut out
the gene to release the cloned genes

Uses of this Technology
• Mass producing proteins
– Human genes like insulin or HGH can be spliced into the plasmid
– The bacteria will then produce the protein
• Improving Plant and Animal Stocks
– Genes can be transferred across species and even plant/animal lines
– Organisms containing these genes are called transgenic organisms

– For example
• Tomatoes have been given resistance to some viruses by inserting certain viral
genes

• Gene Therapy
– Altering a person’s genes to combat disease
• Somatic cell gene therapy
– A patient with melanoma
– Remove a portion of the tumor
– Extract tumor infiltrating lymphocytes (TIL)
– These invade tumors
– Insert a gene that codes for a protein that kills cancer cells in mice into the
TIL’s
– Inject the TIL’s into the patient
– The TIL’s will kill the cancer cells

• Germ cell gene therapy

Polymerase Chain Reaction

• Use the cell’s DNA replicating enzymes to make multiple copies of a piece of DNA
(gene)
• Faster and easier than using plasmids
• DNA is heated and the sides separate
• Short DNA sequences called primers are placed in this mixture-these mark the start
and end of copying
• Taq polymerase (heat resistant) is the enzyme used
• Controls the replication of DNA
• A computer controlled process
• 20 cycles of the process gives in excess of 1 million copies
• Used to make copies of genes