chapter 20. biotechnology: dna technology &...

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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.