Biotechnology Ch 13.4, 15.4, 16 2007-2008

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…

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

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

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

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) https://www.youtube.com/watch?v=sjwNtQYLKeU 7 min

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

Plasmids and Selective Markers https://www.youtube.com/watch?v=5gYYjfa8tdQ Plasmids and Selective Markers 5 min

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)

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 https://www.youtube.com/watch?v=1Q-_qgCtk3c 1 min

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

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.

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. https://www.youtube.com/watch?v=2KoLnIwoZKU 2 min

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 https://www.youtube.com/watch?v=mmgIClg0Y1k 6 min

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.

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?