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

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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

5

5

5

5

5

55

5

5

55

5

5

3

3

3

3

3

3

3

3

3

3

3

3

3

3

3

3

2

3

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

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• 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

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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

DNA Fingerprinting

• DNA fingerprinting

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

5

5

5

3

3

3

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

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Isolate mRNA.

2

1

3

4

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

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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

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Figure 20.19

Mammarycell donor

21

3

4

5

6

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

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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