Post on 27-Jul-2020
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2003-2004AP Biology
Chapter 20.
Biotechnology:DNA Technology & Genomics
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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 & applicationsof biotechnology? direct manipulation of genes for
practical purposes
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Biotechnology The genetic manipulation of organisms
humans have been doing this forthousands of years plant & animal breeding
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Evolution & breeding of food plants
Evolution in morphology of Zea mays from ancestralteosinte (left) to modern corn (right). The middle figureshows possible hybrids of teosinte & early corn varieties
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Evolution & breeding of food plants “Descendants” of the wild mustard
Brassica spp.
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Animal husbandry / breeding
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Biotechnology today Genetic Engineering
manipulation of DNA if you are going to engineer DNA &
genes & organisms, then you need aset of tools to work with
this unit is a surveyof those tools…
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Bioengineering Tool kit Basic Tools
restriction enzymes ligase plasmids / cloning DNA libraries / probes
Advanced Tools PCR DNA sequencing gel electrophoresis Southern blotting microarrays
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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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Cut DNA Restriction enzymes
discovered in 1960s evolved in bacteria to cut up foreign
DNA (“restriction”) protection against viruses & other bacteria
bacteria protect their own DNA by methylation &by not using the recognition sequences
hundreds of different enzymes EcoRI, HindIII, BamHI, SmaI
cut at restriction site specific sequence of DNA symmetrical “palindrome” produces sticky ends
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Discovery of restriction enzymes1960s|1978
Werner Arber Daniel Nathans Hamilton O. Smith
Restriction enzyme movie
Restriction enzymesare named for theorganism they comefrom:EcoRI = 1st restrictionenzyme found in E. coli
Werner Arber discovered restriction enzymes. He postulated thatthese enzymes bind to DNA at specific sites containing recurringstructural elements made up of specific basepair sequences.
Hamilton Smith verified Arber's hypothesis with a purified bacterialrestriction enzyme and showed that this enzyme cuts DNA in themiddle of a specific symmetrical sequence. Other restrictionenzymes have similar properties, but different enzymes recognizedifferent sequences. Ham Smith now works at Celera Genomics, thecompany who sequenced the human genome.
Dan Nathans pioneered the application of restriction enzymes togenetics. He demonstrated their use for the construction of geneticmaps and developed and applied new methodology involvingrestriction enzymes to solve various problems in genetics.
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Adaptive value ofrestriction enzymes?
Blunt vs. sticky ends?
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Paste DNA Sticky ends allow:
H bondsbetweencomplementarybases to anneal
Ligase enzyme “seals”
strands joins sugar-
phosphate bonds
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Copy DNA Plasmids
small, self-replicating circular DNA molecules naturally occur in bacteria insert DNA sequence
into plasmid transformation
insert recombinant plasmidinto bacteria
culture recombinant bacteria= clone of cells
permits production of multiplecopies of a specific gene orDNA sequence
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Recombinant plasmid Antibiotic resistance genes as a selectable marker Restriction sites for
splicing in gene of interest
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Gene cloningRecombinant DNA movie
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Cloning aHuman Gene Use both ampicillin
resistance & color if DNA is correctly
inserted within lacZ genethen colonies will be white intact lacZ gene produces
funtional enzyme: lactose → blue
broken lacZ gene does notproduce functional enzyme lactose ≠ blue
LacZ system was developed to make cloning more efficient.Scientists were frustrated by having to search for bacteria which weresuccessfully transformed with plasmids containing the human gene.LacZ system was developed to quickly distinguish bacteria thatcontained “original” plasmids vs. bacteria that contained plasmidswith the human gene inserted.
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What if you don’t have yourgene conveniently on a chunkof DNA ready to insert into aplasmid?
Have to find your favoritegene (YFG) out of the entiregenome of the organism…
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DNA libraries Cut up genomic DNA
from many cells withrestriction enzyme
Clone all fragmentsinto plasmids at sametime shotgun cloning
Create a storedcollection of genomicDNA fragments
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Find DNA in library Locate YFG
if you know sequence ofprotein… can guess part of DNA
sequence “back translate” protein to
DNA if you have sequence of
similar gene fromanother organism… use part of this sequence
Nucleic acid hybridizationwith probe
Complementationif you have a mutant that lacks YFG, you can transform bacteriawith plasmids from the library until one “cures” (complements)the mutation
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Nucleic acid hybridization identify transformed
bacteria using a probe use tagged
complementary DNAprobe radioactive P32 or
fluorescence heat-treated DNA for
testing = denaturation tounwind strands
DNA hybridizationbetween probe &denatured DNA
Screening the library
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DNA probes Probe
short, single stranded DNA molecule mix with denatured DNA
DNA Hybridization probe bonds to complementary DNA sequence
Label probe is labeled for easy detection
labeled probe
genomic DNA G A T C A G T A G
C T A G T C A T C
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cDNA libraries Collection of only the
coding sequences ofexpressed genes extract mRNA from
cells reverse transcriptase
RNA → DNA from retroviruses
clone into plasmid Applications
need edited DNA forexpression in bacteria human insulin
Could you imagine how much that first insulin clone was worth toGenentech? One little piece of DNA in a plasmid worth billions!It put them on the map & built a multi-billion dollar biotech company.