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Avery, MacLeod, and McCarty1944
• Used bacteria from Griffith’s mouse experiment
• Denatured proteins in membrane and discovered that the DNA still could make other bacteria pathogenic
Biotechnology – pg. 140 in Cliffs Biotechnology – pg. 140 in Cliffs Ch. 20 in textCh. 20 in text
Recombinant DNA – a combination of DNA Recombinant DNA – a combination of DNA segments from two different sourcessegments from two different sources
Can occur through transduction, conjugation, Can occur through transduction, conjugation, transformationtransformation
Can also occur during crossing over during Can also occur during crossing over during meiosis in eukaryotesmeiosis in eukaryotes
Biotechnology – use of biological systems to Biotechnology – use of biological systems to produce products like medicineproduce products like medicine
Often use bacteria and viruses in experiments Often use bacteria and viruses in experiments and production of productsand production of products
Recombinant DNA technologyRecombinant DNA technology Set of lab techniques for combining genes from Set of lab techniques for combining genes from
different sources.different sources. Requires the “cutting” of DNA using restriction Requires the “cutting” of DNA using restriction
enzymesenzymes Restriction enzymes cut DNA at very specific Restriction enzymes cut DNA at very specific
sequences called restriction sitessequences called restriction sites Using bacterial plasmids we can clone specific Using bacterial plasmids we can clone specific
genes to produce proteins of interestgenes to produce proteins of interest Ex. Medicine, farming, oil clean upEx. Medicine, farming, oil clean up
Creating Sticky EndsCreating Sticky Ends
© 2011 Pearson Education, Inc.
Animation: Restriction Enzymes
Right-click slide / select “Play”
Using Restriction Enzymes to cut DNA
• Restriction Enzyme Video
Figure 20.3-3
Recombinant DNA molecule
One possible combinationDNA ligaseseals strands
DNA fragment addedfrom another moleculecut by same enzyme.Base pairing occurs.
Restriction enzymecuts sugar-phosphatebackbones.
Restriction site
DNA5
5
5
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5
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2
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1
Sticky end
GAATTCCTTAAG
CTTAAG AATTC
G
GGAATTC
CTTAA
GG
GG
AATT CAATT CC TTAA C TTAA
Recombinant DNARecombinant DNA
Figure 20.6-5
DNA innucleus
mRNAs incytoplasm
mRNA
Reversetranscriptase Poly-A tail
DNAstrand
Primer
DNA polymerase
cDNA
55
55
55
55
33
33
33
33
A A A A A A
A A A A A A
T T T T T
T T T T T
Figure 20.2 Bacterium
Bacterialchromosome
Plasmid
2
1
3
4
Gene inserted intoplasmid
Cell containing geneof interest
RecombinantDNA (plasmid)
Gene of interest
Plasmid put intobacterial cell
DNA ofchromosome(“foreign” DNA)
Recombinantbacterium
Host cell grown in culture toform a clone of cells containingthe “cloned” gene of interest
Gene of interest
Protein expressed fromgene of interest
Protein harvestedCopies of gene
Basic researchand variousapplications
Basicresearchon protein
Basic research on gene
Gene for pestresistance insertedinto plants
Gene used to alterbacteria for cleaningup toxic waste
Protein dissolvesblood clots in heartattack therapy
Human growthhormone treatsstunted growth
DNA cloningDNA cloning
1.1. Use restriction enzyme to cut a sample of DNA in Use restriction enzyme to cut a sample of DNA in test tube – this will create fragments with sticky test tube – this will create fragments with sticky ends, some will have our gene of interestends, some will have our gene of interest
2.2. Cut a plasmid (cloning vector) with one restriction Cut a plasmid (cloning vector) with one restriction site for the restriction enzyme – the plasmid will site for the restriction enzyme – the plasmid will now have the same sticky ends (plasmid should now have the same sticky ends (plasmid should also be resistant to antibiotic like ampicillin)also be resistant to antibiotic like ampicillin)
3.3. Mix the foreign DNA with the plasmidsMix the foreign DNA with the plasmids
4.4. Apply DNA ligaseApply DNA ligase
Transformation TimeTransformation Time
Place the engineered plasmid into bacterial Place the engineered plasmid into bacterial culture (in test tube)culture (in test tube)
Heat shock and let transformation occurHeat shock and let transformation occur Plate the bacteria and those that grow on Plate the bacteria and those that grow on
ampicillin will have “transformed” with the ampicillin will have “transformed” with the foreign gene of interestforeign gene of interest
Genomic LibraryGenomic Library
At the end, the bacteria now contain our At the end, the bacteria now contain our gene of interest – genomic librarygene of interest – genomic library
Now the gene can be transcribed and Now the gene can be transcribed and translated to make the protein of interesttranslated to make the protein of interest
This DNA is without introns because it was This DNA is without introns because it was made from mRNA using reverse made from mRNA using reverse transcriptase before the experiment. cDNAtranscriptase before the experiment. cDNA
Figure 20.4
Bacterial plasmidTECHNIQUE
RESULTS
ampR gene lacZ gene
Restrictionsite
Hummingbird cell
Sticky ends Gene of
interest
Humming-bird DNAfragments
Recombinant plasmids Nonrecombinant plasmid
Bacteria carryingplasmids
Colony carrying non-recombinant plasmidwith intact lacZ gene
Colony carrying recombinantplasmidwith disruptedlacZ gene
One of manybacterialclones
Figure 20.5
Foreign genome
Cut with restriction enzymes into eithersmallfragments
largefragments
or
Recombinantplasmids
Plasmidclone
(a) Plasmid library
(b) BAC clone
Bacterial artificialchromosome (BAC)
Largeinsertwithmanygenes
(c) Storing genome libraries
Storing Cloned Genes in DNA Libraries
• A genomic library that is made using bacteria is the collection of recombinant vector clones produced by cloning DNA fragments from an entire genome
• A genomic library that is made using bacteriophages is stored as a collection of phage clones
• A clone carrying the gene of interest can be identified with a nucleic acid probe having a sequence complementary to the gene
• This process is called nucleic acid hybridization
© 2011 Pearson Education, Inc.
• A probe can be synthesized that is complementary to the gene of interest
• For example, if the desired gene is
– Then we would synthesize this probe
© 2011 Pearson Education, Inc.
5 3 CTCAT CACCGGC
53 G A G T A G T G G C C G
Figure 20.7
Radioactivelylabeled probemolecules Gene of
interestProbeDNA
Single-strandedDNA fromcell
Film
Location ofDNA with thecomplementarysequence
Nylonmembrane
Nylon membrane
Multiwell platesholding libraryclones
TECHNIQUE 5
53
3
GAGTAGTGGCCG CTCATCACCGGC
Finding specific mutationsGel Electrophoresis
• In humans, researchers analyze the genomes of many people with a certain genetic condition to try to find nucleotide changes specific to the condition
• Genetic markers called SNPs (single nucleotide polymorphisms) occur on average every 100–300 base pairs
© 2011 Pearson Education, Inc.
Figure 20.16
DNA
SNPNormal allele
Disease-causingallele
T
C
Figure 20.10
Normal -globin allele
Sickle-cell mutant -globin allele
Largefragment
Normalallele
Sickle-cellallele
201 bp175 bp
376 bp
(a) DdeI restriction sites in normal andsickle-cell alleles of the -globin gene
(b) Electrophoresis of restrictionfragments from normal andsickle-cell alleles
201 bp175 bp
376 bp
Large fragment
Large fragment
DdeI DdeI DdeI DdeI
DdeI DdeI DdeI
Figure 20.11
DNA restriction enzyme
321
4
TECHNIQUE
I Normal-globinallele
II Sickle-cellallele
III Heterozygote
Restrictionfragments
Nitrocellulosemembrane (blot)
Heavyweight
Gel
Sponge
Alkalinesolution Paper
towels
III III
III III III III
Preparation ofrestriction fragments
Gel electrophoresis DNA transfer (blotting)
Radioactively labeledprobe for -globingene
Nitrocellulose blot
Probe base-pairswith fragments
Fragment from sickle-cell -globin allele
Fragment from normal - globin allele
Filmoverblot
Hybridization with labeled probe Probe detection5
Gel Box
Applications of Gene TechnologyApplications of Gene Technology
DNA FingerprintDNA Fingerprint
Making copies of DNA - PCRMaking copies of DNA - PCR
Polymerase chain reaction (PCR) makes Polymerase chain reaction (PCR) makes copies of DNA in order to have enough copies of DNA in order to have enough sample to run many tests on.sample to run many tests on.
You take the sample of DNA, and heat them You take the sample of DNA, and heat them along with DNA polymerase and A,T,C,G along with DNA polymerase and A,T,C,G “primers”“primers”
They will make millions of copies of the They will make millions of copies of the sample. sample.
Figure 20.8
Genomic DNA
Targetsequence
Denaturation
Annealing
Extension
Primers
Newnucleotides
Cycle 1yields
2molecules
Cycle 2yields
4molecules
Cycle 3yields 8
molecules;2 molecules
(in white boxes)match target
sequence
5
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3
2
3
1
TECHNIQUE
Studying the Expression of Studying the Expression of Interacting Groups of GenesInteracting Groups of Genes
Automation has allowed scientists to measure Automation has allowed scientists to measure the expression of thousands of genes at one the expression of thousands of genes at one time using DNA microarray assaystime using DNA microarray assays
DNA microarray assays DNA microarray assays compare patterns of compare patterns of gene expression in different tissues, at different gene expression in different tissues, at different times, or under different conditionstimes, or under different conditions
© 2011 Pearson Education, Inc.
Isolate mRNA.
2
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TECHNIQUE
Make cDNA by reversetranscription, usingfluorescently labelednucleotides.
Apply the cDNA mixture to a microarray, a different genein each spot. The cDNA hybridizeswith any complementary DNA onthe microarray.
Rinse off excess cDNA; scan microarrayfor fluorescence. Each fluorescent spot(yellow) represents a gene expressedin the tissue sample.
Tissue sample
mRNA molecules
Labeled cDNA molecules(single strands)
DNA fragmentsrepresenting aspecific gene
DNA microarray
DNA microarraywith 2,400human genes
Figure 20.15
Using reverse transcriptase in Using reverse transcriptase in gene therapy gene therapy
Isolate mRNA and use an enzyme called Isolate mRNA and use an enzyme called reverse transcriptase to create DNAreverse transcriptase to create DNA
These artificial DNA molecules can be These artificial DNA molecules can be inserted via a virus into a patient’s cells, inserted via a virus into a patient’s cells, then into the patient.then into the patient.
Gene Therapy in humansGene Therapy in humans
Gene technology in FarmingGene technology in Farming
Golden RiceGolden Rice
Rice injected with DNA Rice injected with DNA that codes for beta-that codes for beta-carotene that we use carotene that we use to make vitamin Ato make vitamin A
DNA injectionDNA injection
Cloning Plants: Single-Cell Cultures
• One experimental approach is to see whether a differentiated cell can generate a whole organism
• A totipotent cell is one that can generate a complete new organism
• Plant cloning is used extensively in agriculture
© 2011 Pearson Education, Inc.
Figure 20.17
Crosssection ofcarrot root
2-mgfragments
Fragments werecultured in nu-trient medium;stirring causedsingle cells toshear off intothe liquid.
Single cellsfree insuspensionbegan todivide.
Embryonicplant developedfrom a culturedsingle cell.
Plantlet wascultured onagar medium.Later it wasplanted in soil.
Adultplant
Reproductive Cloning of Mammals
• In 1997, Scottish researchers announced the birth of Dolly, a lamb cloned from an adult sheep by nuclear transplantation from a differentiated mammary cell
• Dolly’s premature death in 2003, as well as her arthritis, led to speculation that her cells were not as healthy as those of a normal sheep, possibly reflecting incomplete reprogramming of the original transplanted nucleus
© 2011 Pearson Education, Inc.
Figure 20.19
Mammarycell donor
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TECHNIQUE
RESULTS
Culturedmammarycells
Eggcell fromovary
Egg cell donor
NucleusremovedCells fused
Grown in culture
Implanted in uterusof a third sheep
Embryonicdevelopment
Nucleus frommammary cell
Early embryo
Surrogatemother
Lamb (“Dolly”) geneticallyidentical to mammary cell donor
DNA Sequencing
• Relatively short DNA fragments can be sequenced by the dideoxy chain termination method, the first automated method to be employed
• Modified nucleotides called dideoxyribonucleotides (ddNTP) attach to synthesized DNA strands of different lengths
• Each type of ddNTP is tagged with a distinct fluorescent label that identifies the nucleotide at the end of each DNA fragment
• The DNA sequence can be read from the resulting spectrogram
© 2011 Pearson Education, Inc.
Figure 20.12
DNA(template strand)
TECHNIQUE
5
3
C
C
C
C
T
TT
G
G
A
A
AA
GTT
T
DNApolymerase
Primer
5
3
P P P
OH
G
dATP
dCTP
dTTP
dGTP
Deoxyribonucleotides Dideoxyribonucleotides(fluorescently tagged)
P P P
H
G
ddATP
ddCTP
ddTTP
ddGTP
5
3
C
C
C
C
T
TT
G
G
A
A
AA
DNA (templatestrand)
Labeled strands
Shortest Longest5
3
ddCddG
ddAddA
ddA
ddG
ddG
ddTddC
GTT
TGTT
TC
GTT
TC
T T
G
GTT
TCT
GA
GTT
TCT
GAA
GTT
TCT
GAAG
GTT
TCT
GAAGT
GTT
TCT
GAAGTC
GTT
TCT
GAAGTCA
Directionof movementof strands
Longest labeled strand
Detector
LaserShortest labeled strand
RESULTS
Last nucleotideof longestlabeled strand
Last nucleotideof shortestlabeled strand
G
G
G
A
AA
C
C
T
Gene Sequencing
• Sanger Method of Sequencing• DNA sequencing machine ad