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VIRUSES, BACTERIA, andPRIONShttps://www.msu.edu/course/isb/202/ebertmay/images/HIV%20virus.png
http://www.scifair.org/+images/Electrophoresis.gif
http://foodsafety.wisc.edu/assets/foodfacts_2007/wffFeb2007_clip_image002.jpg
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VIRUSES Tiny: smaller than ribosomes
http://academic.pgcc.edu/~kroberts/Lecture/Chapter%2013/13-04_SizesOfViruses_0_L.jpg
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VIRUSES Contain DNA or RNA SINGLE or DOUBLE stranded
NUCLEIC ACID surrounded by PROTEIN coat = CAPSID
Some have ENVELOPE outside capsid
http://micro.magnet.fsu.edu/cells/virus.html
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BACTERIOPHAGES =viruses that infect bacteria
no cellular machinery of their own Can only reproduce in host cells
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HIV (Human Immunodeficiency Virus)AIDS virus RETROVIRUS (Contains RNA) Infects WHITE BLOOD CELLS Has REVERSE TRANSCRIPTASE
Enzyme that can use RNA to make DNA
https://www.msu.edu/course/isb/202/ebertmay/images/HIV%20virus.png
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AP Biology
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PRIONS “Misshaped” proteins Change the shape of other proteins they
contact Aggregates of proteins accumulate in brain
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PRIONS Aggregates of proteins accumulate in brain Neurological disorders
SCRAPIE in sheep BOVINE SPONGIFORM ENCEPHALOPATHY
(BSE) = “Mad Cow” disease CHRONIC WASTING DISEASE
KURU CREUTZFELD-JAKOB
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AP Biology
Bacteria Slide show by Kim Foglia (modified)
Blue edged slides are Kim’s
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AP Biology
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
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AP Biology
Bacterial genome Single circular chromosome
haploid naked DNA
no histone proteins ~4 million base pairs
~4300 genes 1/1000 DNA in eukaryote
How have theselittle guys gotten to
be so diverse??
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AP Biology
Binary fission Replication of bacterial
chromosome Asexual reproduction
offspring genetically identical to parent
where does variation come from?
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AP Biology
Variation in bacteria Sources of variation
spontaneous mutation transformation
plasmids DNA fragments
transduction conjugation transposons
bacteria shedding DNA
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AP Biology
Spontaneous mutation
Spontaneous mutation is a significant source of variation in rapidly reproducing species
Example: E. coli human colon (large intestines) 2 x 1010 (billion) new E. coli each day! spontaneous mutations
for 1 gene, only ~1 mutation in 10 million replications each day, ~2,000 bacteria develop mutation in that
gene but consider all 4300 genes, then:
4300 x 2000 = 9 million mutations per day per human host!
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AP Biology
Transformation 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
promiscuous!?
mix heat-killed pathogenic & non-pathogenicbacteria
mice die
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AP Biology
Plasmids 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 environment
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Genes for antibiotic resistance = R Plasmids Role in rapid evolution Method for spreading “antibiotic resistance”
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AP Biology
Plasmids & antibiotic resistance Resistance is futile?
1st recognized in 1950s in Japan
bacterial dysentery not responding to antibiotics
worldwide problem now resistant genes are
on plasmids that are swapped between bacteria
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AP Biology
TRANSDUCTION with viruses
Phage viruses carry bacterial genes from one host to another
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AP Biology
Conjugation - Bacteria “sex”
Direct transfer of DNA between 2 bacterial cells that are temporarily joined results from presence of F (fertility) plasmid “male” extends sex pilli and attaches to “female”
bacterium cytoplasmic bridge allows transfer of DNA
Animation
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TRANSPOSONS (Transposable elements)
“Jumping” genes Can move from one place to another 1st described by Barbara McClintock in corn Can move genes to new site
http://www.nature.com/nature/journal/v443/n7111/images/443521a-i1.0.jpg
http://www.osti.gov/accomplishments/images/mcclintock_05.jpg
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AP Biology 2007-2008
BiotechnologySlide show by Kim Foglia (modified)
Blue edged slides are Kim’s
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AP Biology
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…
Our tool kit…
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AP Biology
A Brave New World
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• DIAGNOSIS OF DISEASE Virus detection; ID genetic carriers• GENE THERAPY ID mutant genes; purify genes• PHARMACEUTICAL PRODUCTION Bacterial production of insulin, Human Growth hormone, etc• FORENSICS Crime scene analysis• GENETICALLY MODIFIED ORGANISMS “Golden” rice (Vitamin A) Bt-corn-resists insect pests Toxic cleanup bacteria
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AP Biology
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
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AP Biology
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
+
transformedbacteriagene from
other organism
plasmid
cut DNA
recombinantplasmid
vector
glue DNA
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AP Biology
Biotechnology Plasmids used to insert new genes into bacteria
gene we want
cut DNA
cut plasmid DNA
insert “gene we want” into plasmid...
“glue” together
ligase
like what?…insulin…HGH…lactase
Cut DNA?DNA scissors?
recombinant plasmid
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AP Biology
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 bacteriabacteria protect their own DNA by methylation &
by not using the base sequences recognized by the enzymes in their own DNA
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AP Biology
What do you notice about these phrases?
radarracecarMadam I’m AdamAble was I ere I saw Elbaa man, a plan, a canal,
PanamaWas it a bar or a bat I saw?go hang a salami I’m a lasagna
hog
palindromes
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AP Biology
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
Madam I’m Adam
CTGAATTCCGGACTTAAGGC
CTG|AATTCCGGACTTAA|GGC
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AP Biology
Discovery of restriction enzymes1960s | 1978
Werner Arber Daniel Nathans Hamilton O. Smith
Restriction enzymes are named for the organism they come from:EcoRI = 1st restriction enzyme found in E. coli
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AP Biology
RESTRICTION ENDONUCLEASES
• Different enzymes recognize different sequences
• Different kinds of DNA cut with same enzyme will have the same “sticky ends” and can be joined
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AP Biology
Restriction enzymes Cut DNA at specific sites
leave “sticky ends”
GTAACG AATTCACGCTTCATTGCTTAA GTGCGAA
GTAACGAATTCACGCTTCATTGCTTAAGTG
restriction enzyme cut site
restriction enzyme cut site
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AP Biology
Sticky ends Cut other DNA with same enzymes
leave “sticky ends” on both can glue DNA together at “sticky ends”
GTAACG AATTCACGCTTCATTGCTTAA GTGCGAA
gene you want
GGACCTG AATTCCGGATACCTGGACTTAA GGCCTAT
chromosome want to add
gene to
GGACCTG AATTCACGCTTCCTGGACTTAA GTGCGAA
combinedDNA
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AP Biology
Why mix genes together?
TAACGAATTCTACGAATGGTTACATCGCCGAATTCTACG CATTGCTTAAGATGCTTACCAATGTAGCGGCTTAAGATGCTAGC
Gene produces protein in different organism or different individual
aa aaaa aa aa aa aa aa aa aa
“new” protein from organism ex: human insulin from bacteria
human insulin gene in bacteria
bacteria human insulin
How can bacteria read human DNA?
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AP Biology
The code is universal Since all living
organisms… use the same DNA use the same code
book read their genes
the same way
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AP Biology
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
DNA RNA protein trait
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AP Biology
Grow bacteria…make more
growbacteria
harvest (purify)protein
transformedbacteria
plasmid
gene fromother organism
+
recombinantplasmid
vector
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AP Biology
How do you clean up the junk?
reverse transcriptase
Don’t start with DNA… Use mRNA
copy of the gene without the junk!
But in the end, you need DNA to clone into plasmid…
How do you go from RNA DNA? reverse transcriptase from RNA viruses
retroviruses
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REVERSE TRANSCRIPTASE Found in RETROVIRUSES (RNA not DNA) Uses RNA message to make DNA Info flows in reverse RNA → DNA
Can take eukaryotic RNA message after introns have been removed and change it into a DNA sequence to be read by bacteria (no RNA processing in prokaryotes)
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AP Biology
REVERSE TRANSCRIPTASE
http://biology200.gsu.edu/houghton/4564%20'04/figures/lecture%204/AAAreverse.jpg
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AP Biology
Selection for plasmid uptake Antibiotic becomes a selecting agent
only bacteria with the plasmid will grow on antibiotic (ampicillin) plate
LB/amp plateLB plate
all bacteria growonly transformed
bacteria grow
a
aa a
aa
aa
aa
aaa a
a
cloning
a a
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AP Biology
Green with envy??Jelly fish “GFP”
Transformed vertebrates
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Green Fluorescent Protein(GFP)
Genetic tool Originally from
jellyfish Way to tell if gene has
been incorporated
http://www.vet.upenn.edu/schoolresources/communications/publications/bellwether/61/stem_cells.html
http://mabryonline.org/blogs/larkin/GFP%5CGFP_aequorea_victoria-1.jpeg
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AP Biology
Cut, Paste, Copy, Find… Word processing metaphor…
cut restriction enzymes
paste ligase
copy plasmids
bacterial transformation is there an easier way??
find ????
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AP Biology 2007-2008
More Basic Biotechnology ToolsSorting & Copying DNA
Slide show by Kim Foglia (modified)Blue edged slides are Kim’s
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AP Biology
Engineered plasmids
Selectable marker antibiotic resistance
gene on plasmid ampicillin
resistance selecting for
successful transformation successful uptake
of recombinant plasmid
plasmid
ampresistance
restriction sites
EcoRI
BamHI HindIII
Building custom plasmids restriction enzyme sites antibiotic resistance genes as a selectable marker
ori
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AP Biology
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…
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AP Biology
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
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AP Biology
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
+–
DNA
“swimming through Jello”
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AP Biology
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
+–
DNA
“swimming through Jello”
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AP Biology
Gel Electrophoresis
longer fragments
shorter fragments
powersource
completed gel
gel
DNA &restriction enzyme
wells
-
+
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AP Biology
Running a gel
1 2
cut DNA with restriction enzymes
fragments of DNAseparate out based
on size
3
Stain DNA ethidium bromide
binds to DNA fluoresces under
UV light
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AP Biology
Uses: Evolutionary relationships Comparing DNA samples from different
organisms to measure evolutionary relationships
–
+
DNA
1 32 4 5 1 2 3 4 5
turtle snake rat squirrel fruitfly
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AP Biology
Uses: Medical diagnostic Comparing normal allele to disease allele
chromosome with disease-causing
allele 2
chromosomewith normal
allele 1 –
+
allele 1allele 2
DNA
Example: test for Huntington’s disease
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AP Biology
Uses: Forensics Comparing DNA sample from crime
scene with suspects & victim
–
+
S1
DNA
S2 S3 V
suspects crime scene sample
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AP Biology
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
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AP Biology
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
GCTTGTAACGGCCTCATCATCATTCGCCGGCCTACGCTTCGAACATTGCCGGAGTAGTAGTAAGCGGCCGGATGCGAA
GCTTGTAACGGCATCATCATCATCATCATCCGGCCTACGCTTCGAACATTGCCGTAGTAGTAGTAGTAGTAGGCCGGATGCGAA
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AP Biology
Allele 1GCTTGTAACGGCCTCATCATCATTCGCCGGCCTACGCTTCGAACATTGCCGGAGTAGTAGTAAGCGGCCGGATGCGAA
repeats
DNA patterns for DNA fingerprintscut sitescut sites
GCTTGTAACG GCCTCATCATCATCGCCG GCCTACGCTTCGAACATTGCCG GAGTAGTAGTAGCGGCCG GATGCGAA
1 2 3
DNA – +allele 1
Cut the DNA
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AP Biology
Allele 1GCTTGTAACGGCCTCATCATCATTCGCCGGCCTACGCTTCGAACATTGCCGGAGTAGTAGTAAGCGGCCGGATGCGAA
Differences between peoplecut sitescut sites
DNA – +allele 1
Allele 2: more repeats
GCTTGTAACGGCCTCATCATCATCATCATCATCCGGCCTACCGAACATTGCCGGAGTAGTAGTAGTAGTAGTAGGCCGG
DNA fingerprint
allele 2
1 2 3
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AP Biology
RFLPs Restriction Fragment Length Polymorphism
differences in DNA between individuals
change in DNA sequence affects restriction enzyme “cut” site
creates different fragment sizes & different band pattern
Alec Jeffries 1984
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AP Biology
RFLP / electrophoresis use in forensics1st case successfully using DNA evidence
1987 rape case convicting Tommie Lee Andrews
“standard”
“standard”
“standard”
“standard”
semen sample from rapist
semen sample from rapist
blood sample from suspect
blood sample from suspect
How can you compare DNA from
blood & from semen?RBC?
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AP Biology
Electrophoresis use in forensics Evidence from murder trial
Do you think suspect is guilty?
“standard”
blood sample 3 from crime scene
“standard”
blood sample 1 from crime scene
blood sample 2 from crime scene
blood sample from victim 2
blood sample from victim 1
blood sample from suspect OJ Simpson
N Brown
R Goldman
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AP Biology
Uses: Paternity Who’s the father?
+
DNA
childMom F1 F2–
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AP Biology 2007-2008
Making lots of copies of DNA
But it would be so much easier if we didn’t have to use bacteria every time…
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AP Biology
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
No more bacteria,No more plasmids,
No more E. colismelly looks!
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AP Biology
PCR process It’s copying DNA in a test tube! What do you need?
template strand DNA polymerase enzyme nucleotides
ATP, GTP, CTP, TTP primer
Thermocycler
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AP Biology
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
20-30 cycles3 steps/cycle30 sec/step
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AP Biology 2007-2008
http://biology200.gsu.edu/houghton/4564%20'04/figures/lecture%204/pcranimatie.gif
PCR movie
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AP Biology
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)
What does 90°Cdo to our
DNA polymerase?
play DNAi movie
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AP Biology
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
PCR20-30 cycles3 steps/cycle30 sec/step
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AP Biology
Kary Mullis development of PCR technique
a copying machine for DNA
1985 | 1993
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AP Biology 2007-2008
I’m a-glow!
Got any Questions?