3The 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?direct manipulation of genes for practical purposes
4Biotechnology Genetic manipulation of organisms is not new humans have been doing this for thousands of yearsplant & animal breedingINDIRECTLY manipulating DNA
5Evolution & breeding of food plants Evolution of Zea mays from ancestral teosinte (left) to modern corn (right). The middle figure shows possible hybrids of teosinte & early corn varieties
6Evolution & breeding of food plants “Descendants” of the wild mustardBrassica spp.
8Biotechnology today Genetic Engineering Our tool kit… manipulation of DNAif you are going to engineer DNA & genes & organisms, then you need a set of tools to work withthis unit is a survey of those tools…Our tool kit…
11Cut DNA Restriction enzymes restriction endonucleases discovered in 1960sevolved in bacteria to cut up foreign DNA (“restriction”)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
12Restriction enzymes Madam I’m Adam Action of enzyme cut DNA at specific sequencesrestriction sitesymmetrical “palindrome”produces protruding endssticky endsMany different enzymesnamed after organism they are found inEcoRI, HindIII, BamHI, SmaICTGAATTCCGGACTTAAGGCCTG|AATTCCGGACTTAA|GGC
13Discovery of restriction enzymes Werner ArberDaniel NathansHamilton O. SmithRestriction enzymes are named for the organism they come from:EcoRI = 1st restriction enzyme found in E. coliWerner Arber discovered restriction enzymes. He postulated that these enzymes bind to DNA at specific sites containing recurring structural elements made up of specific basepair sequences.Hamilton Smith verified Arber's hypothesis with a purified bacterial restriction enzyme and showed that this enzyme cuts DNA in the middle of a specific symmetrical sequence. Other restriction enzymes have similar properties, but different enzymes recognize different sequences. Ham Smith now works at Celera Genomics, the company who sequenced the human genome.Dan Nathans pioneered the application of restriction enzymes to genetics. He demonstrated their use for the construction of genetic maps and developed and applied new methodology involving restriction enzymes to solve various problems in genetics.Restriction enzyme movie
14Biotech use of restriction enzymes GAATTCCTTAAGDNARestriction enzymecuts the DNASticky ends (complementarysingle-stranded DNA tails)AATTCGAATTCGGCTTAAGCTTAAAdd DNA from another source cut with same restriction enzymeAATTCGGAATTCCTTAAGDNA ligasejoins the strands.Recombinant DNA moleculeGAATTCCTTAAG
15Paste DNA Sticky ends allow: Ligase H bonds between complementary bases to annealLigaseenzyme “seals” strandsbonds sugar-phosphate bondscovalent bond of DNA backbone
16Copy DNA Plasmids small, self-replicating circular DNA molecules insert DNA sequence into plasmidvector = “vehicle” into organismtransformationinsert recombinant plasmid into bacteriabacteria make lots of copies of plasmidgrow recombinant bacteria on agar plateclone of cells = lots of bacteriaproduction of many copies of inserted geneDNA RNA protein trait
17Recombinant plasmid Antibiotic resistance genes as a selectable marker Restriction sites for splicing in gene of interestSelectable markerPlasmid has both “added” gene & antibiotic resistance geneIf bacteria don’t pick up plasmid then die on antibiotic platesIf bacteria pick up plasmid then survive on antibiotic platesselecting for successful transformationselectionHow do we know what’s the right combination of genes on a plasmid?Trail and error research work.selectable markershigh copy rateconvenient restriction sitesThere are companies that still develop plasmids, patent them & sell them.Biotech companies (ex. New England BioLabs)
18Selection for plasmid uptake Ampicillin becomes a selecting agentonly bacteria with the plasmid will grow on amp plateonly transformed bacteria growall bacteria growLB plateLB/amp plate
19Need to screen… Need to make sure bacteria have recombinant plasmid X ampresistanceLacZ generestriction siteslactose blue colorrecombinant plasmidampresistancebroken LacZ genelactose white colorXinserted gene of interestorigin of replicationEcoRIall in LacZ geneBamHIHindIII
20LacZ is a screening system Make sure inserted plasmid is recombinant plasmidLacZ gene on plasmid produces digestive enzymelactose(X-gal) blueblue coloniesinsert foreign DNA into LacZ gene breaks genelactose (X-gal) bluewhite colonieswhite bacterial colonies have recombinant plasmidXXWe want these!!
26What if you don’t have your gene conveniently on a chunk of DNA ready to insert into a plasmid? Have to find your “gene of interest” out of the entire genome of the organism…
27DNA libraries Cut up all of nuclear DNA from many cells of an organism restriction enzymeClone all fragments into plasmids at same time“shotgun” cloningCreate a stored collection of DNA fragmentspetri dish has a collection of all DNA fragments from the organism
28Making a DNA library 1engineered plasmid with selectable marker & screening LacZ geneall DNA from many cells of an organism is cut with restriction enzymesgene of interestall DNA fragments inserted into many plasmidshuman genome = 3 billion basesfragments are cut to ~5000 basestherefore ~ 600,000 fragments per cell.But you have to use many cells to make sure you have a complete set in the library, so… you may have millions of cells that you extracted the DNA from, so… you would need millions of colonies, so the human genome cloned into bacteria would be a walk-in freezer full of petri dishes.clone plasmids into bacteria
29But how do we find colony with our gene of interest in it? Making a DNA library 2recombinant plasmids inserted into bacteriagene of interesthuman genome = 3 billion basesfragments are cut to ~5000 basestherefore ~ 600,000 fragments per cell.But you have to use many cells to make sure you have a complete set in the library, so… you may have millions of cells that you extracted the DNA from, so… you would need millions of colonies, so the human genome cloned into bacteria would be a walk-in freezer full of petri dishes.bacterial colonies (clones) grown on LB/amp/Xgal petri plates
30Find your gene in DNA library Locate Gene of Interestto find your gene you need some of gene’s sequenceif you know sequence of protein…can guess part of DNA sequence“back translate” protein to DNAif you have sequence of similar gene from another organism…use part of this sequenceComplementationif you have a mutant that lacks YFG, you can transform bacteria with plasmids from the library until one “cures” (complements) the mutation?Which bacterial colony has our gene?
31Locating your gene of interest DNA hybridizationfind gene in bacterial colony using a probeshort, single stranded DNA moleculecomplementary to part of gene of interesttagged with radioactive P32 or fluorescenceheat treat genomic DNAunwinds (denatures) strandsDNA hybridization between probe & denatured DNA5’3’labeled probegenomic DNAGATC
32Hybridization 4 1 2 3 Locate Cloning expose film locate colony on plate from filmCloning- plate with bacterial colonies carrying recombinant plasmids1plateplate + filterfilm2Replicate platepress filter paper onto plate to take sample of cells from every colonyHybridization- heat filter paper to denature DNA- wash filter paper with radioactive probe which will only attach to gene of interestfilter3
33Problems… A lot of junk! Introns, introns, introns! introns human genomic library has more “junk” than genes in itIntrons, introns, introns!if you want to clone a human gene into bacteria, you can’t have….introns
34Solution… 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!
35cDNA (copy DNA) libraries Collection of only the coding sequences of expressed genesextract mRNA from cellsreverse transcriptaseRNA DNAfrom retrovirusesclone into plasmidApplicationsneed edited DNA for expression in bacteriahuman insulinCould you imagine how much that first insulin clone was worth to Genentech? One little piece of DNA in a plasmid worth billions! It put them on the map & built a multi-billion dollar biotech company.