BiotechnologyBiotechnologyUsing Biology to Solve Society’s Using Biology to Solve Society’s

ProblemsProblems

13.1 What Is Biotechnology?13.1 What Is Biotechnology?

Genetic engineering – modify DNA to Genetic engineering – modify DNA to produce ______ (?)produce ______ (?) Recombinant DNA – genetically Recombinant DNA – genetically

modified organisms; recombinantmodified organisms; recombinant AgricultureAgriculture Medicines Medicines

Forensics Forensics

Resistant to fungus

13.2 Natural Recombination 13.2 Natural Recombination

Recombination of genes happens Recombination of genes happens naturally in sexually reproducing naturally in sexually reproducing organismsorganisms Meiosis and fertilizationMeiosis and fertilization

Recombination of genesRecombination of genes Law of SegregationLaw of Segregation Law of Independent AssortmentLaw of Independent Assortment Random chanceRandom chance

Crossing overCrossing over

Bacterial RecombinationBacterial Recombination

Bacteria – binary fission (asexual, Bacteria – binary fission (asexual, ‘cloning’)‘cloning’) No genetic variationNo genetic variation

Genetic recombination by 3 means:Genetic recombination by 3 means: Mutation – ‘goof’ in the codeMutation – ‘goof’ in the code Transformation – pick up DNA from Transformation – pick up DNA from

environmentenvironment Transduction – viruses transfer DNA Transduction – viruses transfer DNA Conjugation – bacteria ‘swap’ DNAConjugation – bacteria ‘swap’ DNA

TransformatioTransformationn

Bacteria can absorb DNA from Bacteria can absorb DNA from the environment the environment

Enables some bacteria to Enables some bacteria to become resistant to antibiotics become resistant to antibiotics (MRSA)(MRSA) Griffith’s experiment Griffith’s experiment Non-pathogen became pathogenNon-pathogen became pathogen

plasmid

bacterialchromosome

DNAfragments

bacterialchromosome

Transformation with DNA fragment

Bacterium

bacterialchromosome

Transformation with plasmid

plasmid

Transformation

ConjugationConjugation

Transfer of genes by bacteria that are Transfer of genes by bacteria that are temporarily ‘joined’ temporarily ‘joined’ E. coliE. coli

PiliPili - -‘appendage’ on the surface of a ‘appendage’ on the surface of a bacterium through which cytoplasm can bacterium through which cytoplasm can movemove

PlasmidsPlasmids

Circular DNA in bacteria that carries Circular DNA in bacteria that carries ‘extra’ genes‘extra’ genes F = ‘fertility’; the ability to grow sex piliF = ‘fertility’; the ability to grow sex pili R = various resistance factor(s)R = various resistance factor(s)

TransductiTransductionon

Transfer of genes from Transfer of genes from one bacterium to another one bacterium to another by phagesby phages Hershey-Chase; used T4 Hershey-Chase; used T4

phage to determine that phage to determine that DNA was the molecule of DNA was the molecule of heredityheredity

Restriction EnzymesRestriction Enzymes Bacteria have enzymes that cut DNA Bacteria have enzymes that cut DNA

in in specificspecific locations locations ‘‘Cut out’ phagesCut out’ phages

Enzymes have recognition sites Enzymes have recognition sites Specific sequence of nucleotidesSpecific sequence of nucleotides Palindromes Palindromes

Race car; anna; madam; flee to me Race car; anna; madam; flee to me remote elf; gnu dung; lager sir is remote elf; gnu dung; lager sir is regal; tuna nutregal; tuna nut

Restriction enzymes

BiotechnologyBiotechnology

ForensicsForensics

AgriculturalAgricultural

Medical Medical

Forensics – DNA Forensics – DNA FingerprintingFingerprinting

Restriction enzymes cut pieces Restriction enzymes cut pieces of DNA in very of DNA in very specificspecific locations locations Create pieces of DNA of Create pieces of DNA of

differing lengths differing lengths Restriction Fragment Length Restriction Fragment Length

Polymorphisms, FFLPs Polymorphisms, FFLPs

Other restriction enzymes

ForensicsForensics

RFLPsRFLPs

ForensicsForensics

Large and small pieces can be Large and small pieces can be separated separated

Gel electrophoresisGel electrophoresis – process used to – process used to separate RFLPsseparate RFLPs

1.1. Cut DNA with restriction enzymesCut DNA with restriction enzymes

2.2. DNA ‘loaded’ into a gel bed with wellsDNA ‘loaded’ into a gel bed with wells

3.3. Electricity ‘pulls’ the negative DNA towards Electricity ‘pulls’ the negative DNA towards the positive pole through the gel bedthe positive pole through the gel bed

4.4. Large pieces move slower creating Large pieces move slower creating separationseparation

Gel electrophoresis

DNA samples are pipetted into wells (shallow slots) in the gel. Electrical current is sent through the gel (negative at end with wells, positive at opposite end.)

gel

power supply

wells

pipetter

Electrical current moves DNA segments through the gel. Smaller pieces of DNA move farther toward the positive electrode.

DNA “bands”(not yet visible)

Gel electrophoresis

Southern Blotting TechniqueSouthern Blotting Technique

Too many bands are created during Too many bands are created during electrophoresiselectrophoresis

Have to make specific ‘bands’ visibleHave to make specific ‘bands’ visible Nitrocellulose paper is placed on the gelNitrocellulose paper is placed on the gel Alkaline solution unwinds the DNA helixAlkaline solution unwinds the DNA helix Separated strands of DNA are absorbed Separated strands of DNA are absorbed

by the nitrocellulose paper (blotting)by the nitrocellulose paper (blotting)PCR

Gel is placed on special nylon “paper.” Electrical current drives separatedDNA out of gel onto nylon.

gel

nylon paper

Southern BlottingSouthern Blotting

Radioactive or colored probes are Radioactive or colored probes are mixed with the ‘blot’mixed with the ‘blot’

Probes pair with their Probes pair with their complimentary STRcomplimentary STR

Probes can be seenProbes can be seen

Nylon paper with DNA is bathed in a solution of labeled DNA probes (red) that are complementary to specific DNA segments in the original DNA sample.

nylon paper

solution of DNA probes (red)

STR #1: probe base-pairs and binds

probe label(colored molecule)

STR #2: probe cannot base-pair; does not bind

Complementary DNA segments are labeled by probes (red bands).

DNA ForensicsDNA Forensics STR – short, tandem repeats of STR – short, tandem repeats of

nucleotides (4 or 5) nucleotides (4 or 5) AGATAGATAGATAGATAGATAGATAGATAGATAGATAGAT Common in non-coding regions of Common in non-coding regions of

DNA (introns) DNA (introns) Number of times it repeats is variableNumber of times it repeats is variable DNA fingerprint - use multiple STR’s DNA fingerprint - use multiple STR’s

(10-13) to determine identity(10-13) to determine identity

A

T

G

C

A

T

T

A

A

T

G

C

A

T

T

A

A

T

G

C

A

T

T

A

T

A

A

T

A

T

T

A

T

A

T

A

T

A

G

C

A

T

A

T

G

C

A

T

T

A

8 side-by-side (tandem) repeatsof the same 4-nucleotide sequence,

A

T

G

C

A

T

T

A

A

T

G

C

A

T

T

A

A

T

G

C

A

T

T

A

A

T

G

C

A

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T

A

A

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G

C

A

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A

A

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G

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G

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A

A

T

STR name

Penta D

CSF

D16

D16: an STR on chromosome 16

DNA samples from13 different people

D7

D13

D5

151413121110

98

Num

ber

of r

epea

ts

Variety of STR’s used to isolate one individual

DNA fingerprint

ForensicsForensics

Polymerase Chain Reaction - Polymerase Chain Reaction - amplifies DNAamplifies DNA PCR copies a specific DNA PCR copies a specific DNA

sequencesequence Need to make more copies of the Need to make more copies of the

DNADNADNA is degraded (old)DNA is degraded (old)Not a large enough sampleNot a large enough sample

Each PCR cycle doubles the number of copies of the DNA

1 2 3PCRcycles 4 etc.

1 2 4 8DNAcopies 16 etc.

DNAfragmentto beamplified

One PCR cycle

originalDNA

90 °C 50 °C 72 °C

1 HeatingseparatesDNA strands.

2 Cooling allowsprimers andDNA polymeraseto bind.

3 New DNA strands aresynthesized.

primers DNApolymerase

new DNAstrands

Primers – RNA sequences that tell DNA polymerase where to start copying

Primers + free nucleotides + DNA polymerase + heat = copies of DNA

Copies can be used for forensics, cloning, recombinations

Transgenic OrganismsTransgenic Organisms

Using biotechnology to rearrange Using biotechnology to rearrange genomesgenomes

Medical Medical AgriculturalAgricultural Bioethics Bioethics

Cut both with the same restriction enzyme.

Mix Bt gene and plasmid; add DNA ligase to seal DNA.

Transform Agrobacterium tumifaciens with recombinantplasmid

Infect plant cell with transgenic bacterium.

Insert Bt gene into plant chromosome.

DNA including Bt gene Ti Plasmid

Bt gene

plant cell

plantchromosomes A. tumifaciens

A. tumifaciensplasmids

bacterialchromosome

Cloning a gene

Agricultural Use Agricultural Use

Knowing genome of and organism Knowing genome of and organism enables us to ‘interfere’ with normal enables us to ‘interfere’ with normal protein productionprotein production Insert, remove genes Insert, remove genes

Sterile Insect TechniqueSterile Insect Technique Irradiate then releaseIrradiate then release Insert lethal gene – then releaseInsert lethal gene – then release

Silk worm – industrial strength, glow-Silk worm – industrial strength, glow-in-the-dark silkin-the-dark silk

Transgenic OrganismsTransgenic Organisms

Transgenic mosquitoesTransgenic mosquitoes Anopheles sp. carry malaria parasiteAnopheles sp. carry malaria parasite Insert genes so that they cannot Insert genes so that they cannot

The introduction of genes that impair The introduction of genes that impair PlasmodiumPlasmodium (Malaria parasite)(Malaria parasite) development into mosquito populations is a strategy being development into mosquito populations is a strategy being considered for malaria control. The effect of the transgene on considered for malaria control. The effect of the transgene on mosquito fitness is a crucial parameter influencing the success of mosquito fitness is a crucial parameter influencing the success of this approach. We have previously shown that anopheline this approach. We have previously shown that anopheline mosquitoes expressing the SM1 peptide in the midgut lumen mosquitoes expressing the SM1 peptide in the midgut lumen ((intestineintestine) are impaired for transmission of ) are impaired for transmission of Plasmodium bergheiPlasmodium berghei. . Moreover, the transgenic mosquitoes had no noticeable fitness load Moreover, the transgenic mosquitoes had no noticeable fitness load compared with nontransgenic mosquitoes when fed on noninfected compared with nontransgenic mosquitoes when fed on noninfected mice. Here we show that when fed on mice infected with mice. Here we show that when fed on mice infected with P. bergheiP. berghei, , these transgenic mosquitoes are more fit (higher fecundity and these transgenic mosquitoes are more fit (higher fecundity and lower mortality) than sibling nontransgenic mosquitoes. In cage lower mortality) than sibling nontransgenic mosquitoes. In cage experiments, transgenic mosquitoes gradually replaced experiments, transgenic mosquitoes gradually replaced nontransgenics when mosquitoes were maintained on mice infected nontransgenics when mosquitoes were maintained on mice infected with gametocyte-producing parasites (strain ANKA 2.34) but not with gametocyte-producing parasites (strain ANKA 2.34) but not when maintained on mice infected with gametocyte-deficient when maintained on mice infected with gametocyte-deficient parasites (strain ANKA 2.33). These findings suggest that when parasites (strain ANKA 2.33). These findings suggest that when feeding on feeding on PlasmodiumPlasmodium-infected blood, transgenic malaria-resistant -infected blood, transgenic malaria-resistant mosquitoes have a selective advantage over nontransgenic mosquitoes have a selective advantage over nontransgenic mosquitoes. This fitness advantage has important implications for mosquitoes. This fitness advantage has important implications for devising malaria control strategies by means of genetic modification devising malaria control strategies by means of genetic modification of mosquitoes. of mosquitoes.

Transgenic malaria-resistant mosquitoes have a fitness advantage when feeding on Plasmodium-infected blood

Mauro T. Marrelli, Chaoyang Li, Jason Rasgon, Marcelo Jacobs-Lorena

Dolly and her ewe

Transgenic OrganismsTransgenic Organisms

Restriction enzymes cut bacterial Restriction enzymes cut bacterial plasmid DNA (pDNA)plasmid DNA (pDNA)

Same restriction enzymes cut ‘genes Same restriction enzymes cut ‘genes of interest’ from another organismof interest’ from another organism

‘‘Genes of interest’ added to a Genes of interest’ added to a solution with pDNAsolution with pDNA

DNA ligase (‘glue’) is added to ‘glue’ DNA ligase (‘glue’) is added to ‘glue’ pieces togetherpieces together

Biotechnology In AgricultureBiotechnology In Agriculture

ManyMany Crops Are Genetically Crops Are Genetically ModifiedModified Tomatoes – slow ripening processTomatoes – slow ripening process Tomatoes – insect resistanceTomatoes – insect resistance

Bacillus thuringiensis – Bacillus thuringiensis – produces a natural produces a natural insecticideinsecticide

Desired genes are cloned (insecticide Desired genes are cloned (insecticide gene)gene)

Biotechnology in AgricultureBiotechnology in Agriculture

Transgenic plants are Transgenic plants are cloned cloned in vitroin vitro

Plants are bred together Plants are bred together creating 2creating 2ndnd generation generation plant with insect-plant with insect-resistance gene resistance gene

Plants produce Plants produce antibodies to fight antibodies to fight disease (?)disease (?)

Fish given growth hormones; GMT@

13.5 How Is Biotechnology Used for 13.5 How Is Biotechnology Used for Medical Diagnosis and Treatment?Medical Diagnosis and Treatment?

DNA Technology Can DNA Technology Can Be Used to Diagnose Be Used to Diagnose Inherited DisordersInherited Disorders Restriction Enzymes Restriction Enzymes

May Cut Different May Cut Different Alleles of a Gene at Alleles of a Gene at Different LocationsDifferent Locations

Diagnosing sickle-cell Diagnosing sickle-cell anemia with restriction anemia with restriction enzymes enzymes

Normal allele

Sickle cell

normalglobin allele

Mst II cuts a normal globin allele in 2 places, but cuts thesickle-cell allele in 1 place.

sickle-cellglobin allele

Mst II Mst IIMst II

DNA probe

Mst II Mst II

DNA probe

large

small

AA AS SSHomozygous

Heterozy

gote

Homozygous sickle-cell

Medical TreatmentMedical Treatment

DNA Technology DNA Technology Can Be Used to Can Be Used to Treat DiseaseTreat Disease Examples of Examples of

Currently Used Currently Used Products Produced Products Produced by Recombinant by Recombinant DNA Methods DNA Methods

Medical Diagnosis and TreatmentMedical Diagnosis and Treatment

Treat Cystic FibrosisTreat Cystic Fibrosis Cure Severe Combined Immune Cure Severe Combined Immune

DeficiencyDeficiency

parents with genetic disease

fertilized egg withdefective gene

embryo withgenetic defect

therapeuticgene

genetically correctedcell from culture

egg cellwithoutnucleus

genetically correctedclone of original embryo

healthy baby

baby withgenetic disorder

genetically correctedegg cell

treated culture

viralvector

amniocentesis

vagina

placenta

uterus

fetus

amnioticfluid

chorionic villi

chorionic villus sampling (by suction)

cells: sex determination,biochemical and enzymatic studies

cell culture: biochemical studies, chromosomal analysis, analysis using recombinant DNA methods

centrifugefluid: compositionanalysis

13.6 Ethical Issues of 13.6 Ethical Issues of BiotechnologyBiotechnology

Should Genetically Modified Organisms Should Genetically Modified Organisms Be Permitted in Agriculture?Be Permitted in Agriculture? Are Foods from GMOs Dangerous to Eat?Are Foods from GMOs Dangerous to Eat? Are GMOs Hazardous to the Environment?Are GMOs Hazardous to the Environment?

Should a Human Genome Be Changed by Should a Human Genome Be Changed by Biotechnology?Biotechnology?

Genetic screening

Genetic counseling HGP – the

code

HGP - ethics

HGP - disease