1. 4/19/2018 5:34 PM
Biotechnology
The manipulation of organisms or their components to make useful products.
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2. Biotech Language/Procedures
Genetic Engineering- the direct manipulation of genes for practical purposes.
Recombinant DNA- DNA molecules formed when segments of DNA from two different sources- often different species- are combined in vitro.
Biotechnology has a long history that includes such early practices as selective breeding of farm animals and using microorganisms to make wine and cheese.
Genetic engineering effects everything from agriculture, to criminal law, and medical research.
DNA cloning is used to work with specific genes

3. DNA Cloning
Plasmid- small circular DNA molecule that replicate separately from bacterial chromosome.
Gene Cloning- production of multiple copies of a single gene.
Used to amplify a particular gene or produce a specific protein.
Plasmid has small number of genes that may be useful in specific environments but not necessary for survival or reproduction.

4. Fig. 20-2
Cell containing gene of interest
Bacterium 1
Gene inserted into plasmid
Bacterial chromosome
Plasmid
Gene of interest
Recombinant DNA (plasmid)
DNA of chromosome 2
Plasmid put into bacterial cell
Recombinant bacterium 3
Host cell grown in culture to form a clone of cells containing the “cloned” gene of interest
Gene of Interest
Protein expressed by gene of interest
Copies of gene
Protein harvested
Pg. 397
A common approach to DNA cloning uses E.coli, whose chromosome is a large circular molecule of DNA. In addition, they also have a plasmid. To clone pieces of DNA, the plasmid is first isolated from a bacterial cell and foreign DNA is inserted into it from another source. The resulting plasmid is now a recombinant DNA molecule, combining DNA from two sources. The plasmid is then returned to the bacterial cell, producing a recombinant bacterium. This single cell reproduces through cell division and forms clones of genetically identical cells. Because the dividing bacteria replicate the recombinant plasmid and pass it on to their descendants, the foreign DNA and any genes it carries are cloned at the same time. The production of multiple copies of a single gene is called gene cloning.
Uses of Gene Cloning: to make many copies of a particular gene, and to produce a protein product. If researchers isolate copies of a cloned gene from bacteria, they can use it in basic research, or they can also use it to endow an organism with a new metabolic capability, such as pest resistance. (Ie: a resistance gene present in one crop species might be cloned and transferred into plans of another species. Human growth hormone can also be harvested from cultures of bacteria carrying the gene for this protein, and used in medical purposes). Most protein-coding genes exist in only one copy per chromosome, so the ability to prepare large amounts of that DNA fragment is important. 4
Basic research and various applications
Basic research on gene
Basic research on protein
Gene for pest resistance inserted into plants
Gene used to alter bacteria for cleaning up toxic waste
Protein dissolves blood clots in heart attack therapy
Human growth hor- mone treats stunted growth

5. Restriction Enzymes
Restriction Enzymes- cut DNA molecules at specific locations.
Protect DNA by cutting up foreign DNA from other organisms or phages.
Sticky Ends- single stranded end of restriction fragment.
DNA Ligase- seal sticky ends together.
Each restriction enzyme is very specific and recognizes a particular DNA sequence or “restriction site” and cut both DNA strands at precise points within the restriction site. The DNA of a bacterial cell is protected from the cell’s own restriction enzymes by the addition of methyl groups to adenines or cytosines within the sequences recognized by the enzymes.
(called restriction sites) yielding restriction fragments. Ligase produces stable recombinant molecule.
Most useful restriction enzymes cleave in a staggered manner.
Sticky ends form hydrogen bonds with other complementary sticky ends- how recombinant DNA is formed. Made permanent when sealed by ligase.
Restriction sites are symmetrical- and usually 4-8 nucleotides long.

6. Biotech Techniques
Gel Electrophoresis- a technique for separating nucleic acids or proteins on the basis of their size and electrical charge.
Produces a DNA fingerprint or genetic profile.

7. Techniques
Polymerase Chain Reaction (PCR)- DNA is amplified and copied many times in a test tube.
PCR- billions of copies in a few hours
Relate PCR to what is done on CSI (minute blood and DNA), single embryonic cells for prenatal diagnosis, wooly mammoth (40,000 years old)
PCR cannot substitute for gene cloning in cells when large amounts of a gene are desired. Occasional errors during PCR relication impose limits on the number of good copies that can be made by this method.
Limitation on length of fragment.
Not as reliable as DNA cloning- but if DNA is impure, PCR is more selective. Would take days to clone genes for DNA.

8. Warm Up Exercise
What determines how the DNA fragments separate in gel electrophoresis?
What happens in PCR (polymerase chain reaction)?
What do restriction enzymes do?

9. Detecting Gene Expression
DNA Microarray Assays- detects and measures expression of genes

10. Cloning Plants
Totipotent- a cell that can give rise to all the specialized types of the organism. (plants)

11. Cloning
Nuclear Transplantation- removes the nucleus from an unfertilized egg and replaces it with the nucleus of a differentiated cell.
Mice, cats, cows, horses, pigs, dogs, monkeys.
Do not always look or behave the same. Calico cat different from mother because of random X chromosome inactivation which happens during embryonic development. Just as identical twins are different, so are clones.
Cloned mice are prone to obesity, pneumonia, liver failure, and premature death.

12. Stem Cells
Stem Cells- unspecialized cells that reproduce indefinitely and can differentiate into specialized cells of one or more types.
Embryonic stem cells are considered pluripotent, because they are capable of differentiating into many cell types.
This is the main goal for cloning human embryos- for treating human diseases. Called therapeutic cloning. This is typically considered more ethical than reproductive cloning.

13. Gene Therapy
Gene Therapy- introducing genes into an afflicted individual for therapeutic purposes.

14. Manipulating DNA

15. Terms in vitro Totipotent Pluripotent Stem cell Gene therapy
Transgenic
Genetic profile
Genetically modified
Cloned animals do not always look or behave the same

16. Genetically Modified Products

17. Exit Slip
On a sheet of paper, respond to the question posed at the end of this morning’s reading:
Would you eat meat that came from a cloned animal?
Also, how do you feel about genetically modified agricultural products?
Do you think they are a good thing, and could eliminate some world hunger problems or do you worry that they have been treated with too many antibiotics and are a potential health threat? Provide any additional comments you wish.