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Friday, 12 December 2014

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Kim Foglia & Kelly Reidell

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A Brave New World
Biotechnology today
* Genetic Engineering
* manipulation of DNA
* 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…
* 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
Bacterial genome 
* Single circular chromosome
* haploid
* naked DNA
* no histone proteins
* ~4 million base pairs
* ~4300 genes
* 1/1000 DNA in eukaryote
* Bacteria are opportunists
* pick up naked foreign DNA wherever it may be hanging out
* have surface transport proteins that are specialized for the uptake of naked DNA 
* import bits of chromosomes from other bacteria
* incorporate the DNA bits into their own chromosome
* express new genes
* transformation
* form of recombination
* Small supplemental circles of DNA
* 5000 - 20,000 base pairs
* self-replicating
* carry extra genes
* 2-30 genes 
* genes for antibiotic resistance
* can be exchanged between bacteria
* bacterial sex!!
* rapid evolution 
* can be imported from 
How can plasmids help us?
* A way to get genes into bacteria easily
* insert new gene into plasmid
* insert plasmid into bacteria = vector
* bacteria now expresses new gene
* bacteria make new protein
* Plasmids used to insert new genes into bacteria
How do we cut DNA?
* Restriction enzymes
* restriction endonucleases
* discovered in 1960s
* evolved in bacteria to cut up foreign DNA 
* “restrict” the action of the attacking organism
* protection against viruses 
& other bacteria
* bacteria protect their own DNA by methylation & by not using the base 
sequences recognized 
by the enzymes 
in their own DNA
What do you notice about these phrases?
Restriction enzymes
* Action of enzyme 
* cut DNA at specific sequences
* restriction site
* symmetrical “palindrome”
* produces protruding ends
* sticky ends
* will bind to any complementary DNA
* Many different enzymes
* named after organism they are found in
* EcoRI, HindIII, BamHI, SmaI
Restriction enzymes
* Cut DNA at specific sites
* leave “sticky ends”
Sticky ends
* Cut other DNA with same enzymes
* leave “sticky ends” on both
* can glue DNA together at “sticky ends”

Sticky ends help glue genes together
Why mix genes together?
* Gene produces protein in different organism or different individual
The code is universal
* Since all living organisms… 
* use the same DNA
* use the same code book
* read their genes the same way
Copy (& Read) DNA
* Transformation
* insert recombinant plasmid 
into bacteria
* grow recombinant bacteria in agar cultures 
* bacteria make lots of copies of plasmid
* “cloning” the plasmid
* production of many copies of inserted gene
* production of “new” protein
* transformed phenotype
Grow bacteria…make more
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
Green with envy??
Cut, Paste, Copy, Find…
* Word processing metaphor…
* cut
* restriction enzymes
* paste
* ligase
* copy
* plasmids
* bacterial transformation
* is there an easier way??
* find
* ????

More Basic Biotechnology Tools
Sorting & Copying DNA
Many uses of restriction enzymes…
* Now that we can cut DNA with restriction enzymes…
* we can cut up DNA from different people… or different organisms… 
and compare it
* why?
* forensics
* medical diagnostics
* paternity
* evolutionary relationships 
* and more…
Comparing cut up DNA
* How do we compare DNA fragments?
* separate fragments by size
* How do we separate DNA fragments?
* run it through a gelatin
* agarose
* made from algae 
* gel electrophoresis
Gel electrophoresis
* A method of separating DNA in a gelatin-like material using an electrical field
* DNA is negatively charged
* when it’s in an electrical field it moves toward the positive side  
Gel electrophoresis
* DNA moves in an electrical field…
* so how does that help you compare DNA fragments?
* size of DNA fragment affects how far it travels
* small pieces travel farther
* large pieces travel slower & lag behind
Gel Electrophoresis
Running a gel
Stain DNA
* ethidium bromide binds to DNA
* fluoresces under UV light
Uses: Evolutionary relationships
* Comparing DNA samples from different organisms to measure evolutionary relationships
Uses: Medical diagnostic
* Comparing normal allele to disease allele
Uses: Forensics
* Comparing DNA sample from crime scene with suspects & victim
DNA fingerprints
* Comparing blood samples on defendant’s clothing to determine if it belongs to victim
* DNA fingerprinting
* comparing DNA banding pattern between different individuals
* ~unique patterns
Differences at the DNA level
* Why is each person’s DNA pattern different?
* sections of “junk” DNA
* doesn’t code for proteins
* made up of repeated patterns
* CAT, GCC, and others
* each person may have different number of repeats
* many sites on our 23 chromosomes with 
different repeat patterns
DNA patterns for DNA fingerprints
Differences between people
* Restriction Fragment Length Polymorphism
* differences in DNA between individuals
Polymorphisms in populations
* Differences between individuals at 
the DNA level
* many differences accumulate in “junk” DNA
RFLP / electrophoresis use in forensics
* 1st case successfully using DNA evidence
* 1987 rape case convicting Tommie Lee Andrews
Electrophoresis use in forensics
* Evidence from murder trial
* Do you think suspect is guilty?
Uses: Paternity 
* Who’s the father?
Making lots of copies of DNA
But it would be so much easier if we didn’t have to use bacteria every time…
Copy DNA without plasmids? PCR!
* Polymerase Chain Reaction
* method for making many, many copies of a specific segment of DNA
* ~only need 1 cell of DNA to start
PCR process
* It’s copying DNA in a test tube!
* What do you need?
* template strand
* DNA polymerase enzyme
* nucleotides
* primer 
PCR primers
* The primers are critical!
* need to know a bit of sequence to make proper primers
* primers can bracket target sequence
* start with long piece of DNA & copy a specified shorter segment
* primers define section of DNA to be cloned
PCR process
* What do you need to do?
* in tube: DNA, DNA polymerase enzyme, primer, nucleotides 
* denature DNA: heat (90°C) DNA to separate strands
* anneal DNA: cool to hybridize with primers & build DNA (extension)
The polymerase problem
* Heat DNA to denature (unwind) it
* 90°C destroys DNA polymerase
* have to add new enzyme every cycle
* almost impractical!
* Need enzyme that can 
withstand 90°C…
* Taq polymerase
* from hot springs bacteria
* Thermus aquaticus


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