ap biology 2005-2006 chapter 6 biotechnology: dna technology & genomics 1
TRANSCRIPT
AP Biology 2005-2006
Chapter 6
Biotechnology:DNA Technology & Genomics
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AP Biology 2005-2006
The BIG Questions… How can we use our knowledge of DNA to:
diagnose disease or defect? cure disease or defect? change/improve organisms?
What are the techniques & applications of biotechnology?
Biotechnology The use of living organisms (or substances
from living organisms) for practical purposes
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Biotechnology Use of organisms to create a
desired product is not new humans have been doing
this for thousands of years Alcohol fermentation (for
brewing beer and food preservation)
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Biotechnology Agriculture
Plant and animal breeding Selective breeding – altered the genomes of
crops by breeding them with other plants.
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Biotechnology today Genetic Engineering
Direct manipulation of DNA if you are going to engineer DNA &
genes & organisms, then you need a set of tools to work with
this chapter is a survey of those tools…
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Bioengineering Tool kit Basic Tools
restriction enzymes ligase plasmids / cloning DNA databases / probes
Advanced Tools PCR DNA sequencing gel electrophoresis Southern blotting microarrays
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Isolating DNABefore DNA can be manipulated, it needs to be
isolated from cells.
1. 1. Cell membranes are disrupted use a detergent Salt used to neutralize lipids
2. 2. DNA precipitation ethanol used to dehydrate and aggregate DNA Salt used to neutralize phosphate groups in DNA
3. DNA isolation / storage
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DNA Isolation
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1. Cut DNA To study the functions
of individual genes, molecular biologists will cut desired genes out of
a genomeplace the gene into
bacterial plasmidproduce recombinant
DNA
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1. Cut DNA treat isolated DNA
with restriction enzymes restriction
endonucleases Evolved in bacteria
Immune System protection against
viruses & other bacteria
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Restriction enzymes - Action cut DNA at specific sequences
Restriction (recognition) site Are usually 4-8 bp in length
produces protruding ends sticky ends that contain DNA
nucleotides that lack complementary bases
Some do not produce protruding ends blunt ends
CTGAATTCCGGACTTAAGGC
CTG|AATTCCGGACTTAA|GGC
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Restriction Enzymes
Many different enzymes named after
organism they are found in EcoRI,
HindIII, BamHI, SmaI
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Restriction Enzyme Cutting
sticky ends – enzyme digests (cuts) to make overhangs
EcoRI 5’ G A A T T C 3’3’ C T T A A G 5’
5’ G 3’ 5’ A A T T C 3’
3’ C T T A A 5’ 3’ G 5’
5’ overhang
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Restriction Enzyme Cutting
PstI 5’ C T G C A G 3’3’ G A C G T C 5’
5’ C T G C A 3’ 5’ G 3’3’ G 5’ 3’ A C G T C 5’
3’ overhang
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2. Paste DNA Sticky ends allow:
H bonds between complementary bases to anneal
DNA Ligase enzyme “seals”
strands bonds sugar-
phosphate backbone together
Condensation reaction
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DNA Ligase T4 DNA ligase
originated in T4 bacteriophages
used to chemically join two blunt ends of DNA together
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AATTC
AATTC
AATTC
GAATTC
G
G
GG
G
GAATTC
CTTAAG
GAATTCCTTAAG
CTTAA
CTTAA
CTTAAG
DNA ligasejoins the strands.
DNA
Sticky ends (complementarysingle-stranded DNA tails)
Recombinant DNA molecule
AATTCGG
CTTAA
Biotech use of restriction enzymes
Restriction enzymecuts the DNA
Add DNA from another source cut with same restriction enzyme
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Exercise 1
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Cut, Paste, Copy, Find… Word processing metaphor…
1. cut Isolate desired DNA restriction enzymes
2. paste ligase
3. copy plasmids
bacteria transformation
PCR 4. find
Southern blotting / probes 19
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Remember… Prokaryotic genomes (e.g. bacteria)
contain Chromosome Plasmids
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Plasmid
Chromosome
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Plasmid pBR322
Plasmids Contain “accessory
genes” Antibiotic resistance
Can carry and express foreign genes Plasmids are vectors
Vehicles by which DNA can be introduced into host cells
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Plasmid Restriction Maps
Shows the location of cleavage sites for many different enzymes These maps are used
like road maps to the DNA molecule
Numbers beside restriction enzyme indicates at which base pair the DNA is cut by that particular enzyme
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Sample Problem: Determine the size and
number of fragments that would be produced if the plasmid was digested with: EcoRV and Pvu II?
2 cuts, therefore 2 fragments
Between sites:
2064-185=1879 bp Rest:
4361-1879=2482 bp
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Why bacteria? Plasmid uptake is easy Cheap Reproduce rapidly and
frequently Produce multiple
copies of the recombinant DNA and protein in a short amount of time Recombinant
DNA and proteins are kept in large storage database to be used later
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Bacterium
Bacterialchromosome
Plasmid
Cell containing geneof interest
RecombinantDNA (plasmid)
Gene of interest
DNA ofchromosome
Recombinatebacterium
Protein harvested
Basic research on protein
Gene of interest
Copies of gene
Basic research on gene
Gene for pestresistance inserted
into plants
Gene used to alterbacteria for cleaning
up toxic waste
Protein dissolvesblood clots in heart
attack therapy
Human growth hormone treatsstunted growth
Protein expressedby gene of interest
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Protein Databases
Actin Protein
Unnamed Protein
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Review: Transformation vs. Recombination
Transformation the introduction of foreign DNA (usually
a plasmid) into a bacterial cell
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Recombination Fragment of DNA
composed of sequences originating from at least two different sources
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3. Copying (Cloning)
1 Isolate plasmid DNA and human DNA.
2 Cut both DNA samples with the same restriction enzyme
3 Mix the DNAs; they join by base pairing. The products are recombinant plasmids andmany nonrecombinant plasmids.
TECHNIQUE In this example, a human gene is inserted into a plasmid from E. coli. The plasmid contains the ampR gene, which makes E. coli cells resistant to the antibiotic ampicillin. It also contains the lacZ gene, which encodes -galactosidase. This enzyme hydrolyzes a molecular mimic of lactose (X-gal) to form a blue product. Only three plasmids and three human DNA fragments are shown, but millions of copies of the plasmid and a mixture of millions of different human DNA fragments would be present in the samples.
Stickyends Human DNA
fragments
Human cell
Gene of interest
Bacterial cell
ampR gene(ampicillinresistance)
Bacterial plasmid
Restriction site
Recombinant DNA plasmids
lacZ gene (lactose breakdown)
Figure 20.4
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RESULTS Only a cell that took up a plasmid, which has the ampR gene, will reproduce and form a colony. Colonies with nonrecombinant plasmids will be blue, because they can hydrolyze X-gal. Colonies with recombinant plasmids, in which lacZ is disrupted, will be white, because they cannot hydrolyze X-gal. By screening the white colonies with a nucleic acid probe (see Figure 20.5), researchers can identify clones of bacterial cells carrying the gene of interest.
Colony carrying non-recombinant plasmid with intact lacZ gene
Bacterialclone
Colony carryingrecombinant plasmidwith disrupted lacZ gene
Recombinantbacteria
4Introduce the DNA into bacterial cells (transoformation).
5 Plate the bacteria on agar containingampicillin and X-gal (lactose). Incubate untilcolonies grow.
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How do we know if it worked? We know cloning worked if:
Transformation has occurred i.e. there has been plasmid
uptake into the bacteria Recombination has
occurred i.e. foreign genes have been
inserted into the bacterial plasmid
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Selecting for successful transformation
Antibiotic resistance genes as a selectable marker
Plasmid has both “added” gene & antibiotic resistance gene
selection
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Amp SelectionIf bacteria don’t pick up
plasmidthey will not have
antibiotic resistancedie on antibiotic (amp)
platesIf bacteria pick up
plasmid survive on antibiotic
(amp) plates
We have selected only those colonies that have undergone successful transformation
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Amp Selection
Ampicillin is the selecting agent
LB/amp plateLB plate
all bacteria grow
only transformed bacteria grow
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Screening for successful recombination
Transformed colonies have both
recombinant and non-recombinant plasmids
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recombinant non-recombinant
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LacZ Screening
LacZ gene codes for β galactosidase
Lactose (or X-gal) is our screening agent
plasmid
ampresistance
LacZ gene
restriction sites
lactose blue color
recombinantplasmid
ampresistance
brokenLacZ gene
lactose white colorX
insertedgeneof interest
origin ofreplication
all in LacZ geneEcoRIBamHI
HindIII
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LacZ screening system
XX Make sure inserted plasmid is
recombinant plasmid LacZ gene on plasmid
produces digestive enzyme lactose(X-gal) blue blue colonies
insert foreign DNA into LacZ gene breaks gene lactose (X-gal) blue white colonies
white bacterial colonies have recombinant plasmid
We want
these
!!
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Amp selection & LacZ screening
- gene of interest
- LacZ gene
- amp resistance
LB/amp LB/amp/Xgal
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Cut, Paste, Copy, Find… Word processing metaphor…
cut restriction enzymes
paste ligase
copy plasmids
bacteria transformation
PCR find
Southern blotting / probes
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4. Find White colonies
could have recombinant plasmids desired and
undesired genes
How do you find the conony with the gene of interest?
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4. Find
recombinant plasmids inserted into bacteria
gene of interest
bacterial colonies (clones) grown on LB/amp/Xgal petri plates
But howdo we find
colony with our gene of interest
in it?
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DNA hybridization find gene in bacterial colony using a probe
short, single stranded DNA molecule complementary to part of gene of interest tagged with radioactive P32 or fluorescence
heat treat genomic DNA unwinds (denatures) strands
DNA hybridization between probe & denatured DNA
Locating your gene of interest
5’3’
labeled probe
genomic DNAG A T C A G T A G
C T A G T C A T C
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HybridizationCloning- plate with bacterial
colonies carrying recombinant plasmids
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2
Hybridization- heat filter paper to
denature DNA- wash filter paper with
radioactive probe which will only attach to gene of interest
Replicate plate- press filter paper onto
plate to take sample of cells from every colony
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Locate- expose film- locate colony on plate
from film
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film
filter
plate
plate + filter
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Classwork/Homework Pg. 281 #1-3 Pg. 282 #6,7,10 Pg. 287 #16,18 Pg. 289 #20,21 Pg. 291 #2-7,10,11(b,c),15,16(a,b),17,19 Pg. 295 #4,5
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