1. Biotechnology Ch 13.4, 15.4, 16

2. Biotechnology today Our tool kit…
Biotechnology- manipulation of organisms or their components to perform practical tasks or provide useful products
Genetic Engineering
manipulation of DNA (recombinant DNA are molecules formed when segments of DNA from 2 different sources are combined)
if you are going to engineer DNA & genes & organisms, then you need a set of tools to work with
this unit is a survey of those tools…
Our tool kit…

3. Bacteria Bacteria review one-celled prokaryotes reproduce by mitosis
binary fission
rapid growth
generation every ~20 minutes
108 (100 million) colony overnight!
dominant form of life on Earth
incredibly diverse
Plasmids can be imported from environment

4. How can plasmids help us?
A way to get genes into bacteria easily
insert new gene into plasmid
insert plasmid into bacteria = vector (is a plasmid or a virus used to transfer foreign genetic material into a cell)
bacteria now expresses new gene
bacteria make new protein
transformed bacteria
gene from other organism
recombinant plasmid
vector
plasmid
cut DNA +
glue DNA

5. Restriction enzymes  CTGAATTCCG GACTTAAGGC  CTG|AATTCCG GACTTAA|GGC
restriction endonucleases
evolved in bacteria to cut up foreign DNA
“restrict” the action of the attacking organism
protection against viruses & other bacteria
Action of enzyme
cut DNA at specific sequences
restriction site
produces protruding ends
sticky ends
will bind to any complementary DNA
Via DNA ligase
Many different enzymes
named after organism they are found in
EcoRI (1st restriction enzyme found in E. coli) 
CTGAATTCCG
GACTTAAGGC 
CTG|AATTCCG
GACTTAA|GGC

7. Plasmids continued
GFP- discovered in jelly fish, used to identify recombinant genes, Green Flourescent Protein.
Electroporation- zap with electricity
Use calcium chloride to heat shock bacteria to change their cell walls (easier to pick up)
7 min

8. Storing Cloned Genes in DNA libraries
Genomic library- is the collection of recombinant vector clones produced by cloning DNA fragments from an entire genome; petri dish with all DNA fragments

9. Plasmids and Selective Markers
Plasmids and Selective Markers
5 min

10. Making cDNA (complementary DNA) for a eukaryotic gene
Flowing backwards so enzymes can take edited messages and change it into a gene
Reverse transcriptase from retroviruses (RNA containing virus)

11. https://www.youtube.com/watch?v=1Q-_qgCtk3c 1 min
DNA Analysis
Gel electrophoresis: separates nucleic acids or proteins on the basis of size or electrical charge creating DNA bands of the same length
Southern Blot- uses radioactively labeled probes to ID specific DNA segments, expose xray film
1 min

12. Restriction fragment analysis
Based on the fact that the amount of "junk DNA" differs uniquely between individuals; Structural genes are often separated by large regions of repeating base pairs.
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 FINGERPRINT
Restriction fragment length polymorphisms (RFLPs)- technique that exploits variations in DNA sequences

13. 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.

14. Polymerase chain reaction (PCR)
Amplification of any piece of DNA without cells (in vitro)
Materials: heat (unwinds DNA), DNA polymerase (Taq polymerase), nucleotides, single-stranded DNA primers
Applications: fossils, forensics, prenatal diagnosis, etc.
2 min

15. https://www.youtube.com/watch?v=mmgIClg0Y1k 6 min
DNA Sequencing
Determination of nucleotide sequences
Genomics: the study of genomes based on DNA sequences
Human Genome Project
6 min

16. Uses of genetic engineering
Genetically modified organisms (GMO)
enabling plants to produce new proteins
Protect crops from insects: BT corn
corn produces a bacterial toxin that kills corn borer (caterpillar pest of corn)
Extend growing season: fishberries
strawberries with an anti-freezing gene from flounder
Improve quality of food: golden rice
rice producing vitamin A improves nutritional value
For example, a transgenic rice plant has been developed that produces yellow grains containing beta-carotene.
Humans use beta-carotene to make vitamin A.
Currently, 70% of children under the age of 5 in Southeast Asia are deficient in vitamin A, leading to vision impairment and increased disease rates.

17. Practical DNA Technology Uses
Diagnosis of disease (virus detection)
Human gene therapy
Pharmaceutical products (vaccines, insulin, human growth hormone)
Forensics (paternity, crime scene analysis)
Animal husbandry (transgenic organisms)
Genetic engineering in plants
Ethical concerns?