Genetic Engineering Genetic Engineering and Biotechnologyand Biotechnology

IB Biology HL IIB Biology HL I

Spring 2014Spring 2014

Mrs. PetersMrs. Peters

What do we already What do we already know?know?

What does DNA contain?What does DNA contain? What is the function of a gene?What is the function of a gene? What do you need to make a What do you need to make a

protein?protein? How are prokaryotes and eukaryotes How are prokaryotes and eukaryotes

different?different?

What is Genetic What is Genetic Engineering?Engineering?

Genetic Engineering:Genetic Engineering: the direct the direct manipulation of DNA for practical manipulation of DNA for practical purposespurposes used in Forensic Science, agriculture, used in Forensic Science, agriculture,

medicine and food technologymedicine and food technology

Biotechnology:Biotechnology: the manipulation the manipulation (through genetic engineering) of (through genetic engineering) of living organisms or their living organisms or their components to produce useful components to produce useful usually commercial productsusually commercial products

Important TermsImportant Terms Gene CloningGene Cloning: making multiple : making multiple

identical copies of a gene (specific identical copies of a gene (specific pieces of DNA)pieces of DNA)

CloneClone: a group of genetically identical : a group of genetically identical organisms or a group of cells derived organisms or a group of cells derived from a single parent cell.from a single parent cell.

Plasmid:Plasmid: circular DNA found in bacteria, not part of the nucleoid region

Restriction EnzymesRestriction Enzymes: enzymes that : enzymes that protect bacteria by cutting up foreign protect bacteria by cutting up foreign DNA from invadersDNA from invaders

DNA ligaseDNA ligase: enzyme used to seal DNA : enzyme used to seal DNA strands togetherstrands together

Important TermsImportant Terms Restriction siteRestriction site: the same sequence of : the same sequence of

4 to 8 nucleotides, usually symmetrical, 4 to 8 nucleotides, usually symmetrical, where restriction enzymes cut DNAwhere restriction enzymes cut DNA

Restriction Fragment:Restriction Fragment: the piece of the piece of DNA that is cut out of a DNA strand by DNA that is cut out of a DNA strand by restriction enzymes.restriction enzymes.

Sticky endsSticky ends: short single stranded : short single stranded sequences on both sides of a restriction sequences on both sides of a restriction fragment of DNAfragment of DNA

Recombinant DNA:Recombinant DNA: a “new” DNA a “new” DNA strand which contains the original DNA strand which contains the original DNA + a restriction fragment. (recombined + a restriction fragment. (recombined DNA)DNA)

Restriction EnzymesRestriction Enzymes

Enzymes that cut DNA molecules at Enzymes that cut DNA molecules at specific locationsspecific locations

Discovered in late 1960’sDiscovered in late 1960’s Found in Prokaryotes, used naturally Found in Prokaryotes, used naturally

to protect bacteria from invading to protect bacteria from invading DNA from other organismsDNA from other organisms

Used in science to make DNA Used in science to make DNA fragments and recombinant DNAfragments and recombinant DNA

Making Recombinant Making Recombinant DNADNA

1. 1. Restriction enzymes Restriction enzymes recognize a specific DNA recognize a specific DNA sequencesequence

2. Restriction enzyme cuts 2. Restriction enzyme cuts DNA, producing sticky DNA, producing sticky endsends

3. DNA fragment from other 3. DNA fragment from other source is added; fragments source is added; fragments stick together by base stick together by base pairingpairing

4. DNA ligase seals the 4. DNA ligase seals the strands, resulting in strands, resulting in recombinant DNA recombinant DNA moleculemolecule

Time to Practice!Time to Practice!

RFLP Activity-DNA ScissorsRFLP Activity-DNA Scissors

DNA CloningDNA Cloning

Process of making Process of making multiple identical multiple identical copies of a gene copies of a gene aka gene cloningaka gene cloning

DNA technology DNA technology uses bacteria uses bacteria plasmids to clone plasmids to clone genesgenes

DNA and Gene Cloning DNA and Gene Cloning ProcessProcess

1.1. Isolate plasmid DNA Isolate plasmid DNA from bacteria and from bacteria and DNA gene from other DNA gene from other organismorganism

2.2. Using a restriction Using a restriction enzyme, cut the enzyme, cut the plasmid and the gene plasmid and the gene from the other DNA from the other DNA strandstrand

3.3. Gene is inserted into Gene is inserted into plasmid and sealed plasmid and sealed with DNA Ligase, with DNA Ligase, forming recombinant forming recombinant DNADNA

DNA and Gene Cloning DNA and Gene Cloning ProcessProcess

4. Plasmid put into 4. Plasmid put into bacterial cellbacterial cell

5. Cell grown in culture, 5. Cell grown in culture, forming clones (lots of forming clones (lots of copies)copies)

6. Desired gene is 6. Desired gene is identified by products identified by products formed (protein or formed (protein or characteristic)characteristic)

Applications of CloningApplications of Cloning Copies of Genes:Copies of Genes:

For pest resistance in For pest resistance in plantsplants

For basic research on For basic research on genesgenes

To alter bacteria for To alter bacteria for cleaning toxic wastecleaning toxic waste

Copies of proteins:Copies of proteins: Human growth hormone Human growth hormone

treatmentstreatments For basic research on For basic research on

proteinsproteins Dissolving blood clots in Dissolving blood clots in

heart attack therapyheart attack therapy

Time to PracticeTime to Practice

Plasmid SimulationPlasmid Simulation

DNA ProfilingDNA Profiling DNA profiling is the process of DNA profiling is the process of

matching DNA from a collected matching DNA from a collected sample to a known individual.sample to a known individual.

DNA samples come from hair, skin, DNA samples come from hair, skin, blood and other body fluidsblood and other body fluids

DNA profiling is used in forensic DNA profiling is used in forensic science to establish the possibility of science to establish the possibility of guilt or prove a suspect innocent; also guilt or prove a suspect innocent; also used in paternity testingused in paternity testing

What happens on CSI and NCISWhat happens on CSI and NCIS

PCRPCR

PPolymerase olymerase CChain hain RReaction eaction a technique in a technique in

which a specific which a specific piece of DNA is piece of DNA is copied quickly copied quickly without the use of without the use of cellscells

Used for DNA Used for DNA profiling in forensic profiling in forensic science and science and paternity testingpaternity testing

PCR: What is the machine PCR: What is the machine doing?doing?

PCR Process:PCR Process: DNA is incubated in DNA is incubated in

a test tube with a test tube with DNA polymeraseDNA polymerase, , nucleotides and nucleotides and short pieces of short pieces of synthetic single-synthetic single-stranded DNAstranded DNA to act to act as primers for DNA as primers for DNA synthesissynthesis

This is a three-cycle This is a three-cycle process which process which continues until the continues until the targeted sequence targeted sequence has been duplicated has been duplicated many times.many times.

PCR: What is the machine PCR: What is the machine doing?doing?

PCR Process:PCR Process:1.1. Heat briefly to Heat briefly to

separate DNA strandsseparate DNA strands2.2. Cool to allow primers Cool to allow primers

to hydrogen bondto hydrogen bond3.3. DNA polymerase adds DNA polymerase adds

nucleotides to the 3’ nucleotides to the 3’ end of each primerend of each primer

4.4. After 1 cycle, 2 DNA After 1 cycle, 2 DNA molecules are mademolecules are made

5.5. Process repeats, each Process repeats, each cycle takes about 5 cycle takes about 5 minutes and doubles minutes and doubles the targeted DNA the targeted DNA sequence each time.sequence each time.

PCR: What is the machine PCR: What is the machine doing?doing?

PCR End Result: PCR End Result: millions of millions of identical DNA identical DNA fragments fragments

These copies are used These copies are used to produce a DNA to produce a DNA profile which can profile which can be used in crime be used in crime scene analysis and scene analysis and paternity testing.paternity testing.

DNA Analysis DNA Analysis Gel electrophoresisGel electrophoresis

Technique used to Technique used to separate macromolecules separate macromolecules based on size or based on size or electrical chargeelectrical charge

Sorts DNA fragments by Sorts DNA fragments by size in bands containing size in bands containing molecules of the same molecules of the same lengthlength

Restriction fragment Restriction fragment analysis detects DNA analysis detects DNA differences differences Use restriction enzymes Use restriction enzymes

to cut DNA and to cut DNA and electrophoresis to electrophoresis to separate fragmentsseparate fragments

Gel Electrophoresis Gel Electrophoresis ProcessProcess

1.1. Samples are placed in wells at one end of the gelSamples are placed in wells at one end of the gel2.2. Electrodes are attached to electrophoresis Electrodes are attached to electrophoresis

chamberchamber3.3. Molecules migrate toward the opposite pole, Molecules migrate toward the opposite pole,

depending on chargedepending on charge4.4. When current is turned off, molecules are arrayed When current is turned off, molecules are arrayed

in bands along a lane according to sizein bands along a lane according to size

Uses of ElectrophoresisUses of Electrophoresis DNA Profiling for DNA Profiling for

Paternity TestsPaternity Tests Forensic Forensic

investigationinvestigation DNA AnalysisDNA Analysis Human Genome Human Genome

ProjectProject

Time to Practice!Time to Practice!

Electrophoresis Simulation: DNA Electrophoresis Simulation: DNA Goes to the RacesGoes to the Races

Human Genome ProjectHuman Genome Project In 1990, the Human Genome In 1990, the Human Genome

Project started as an Project started as an international collaboration international collaboration to determine the entire base to determine the entire base sequence of the human sequence of the human genome.genome.

Public research groups Public research groups (universities) and private (universities) and private companies (Celera) worked companies (Celera) worked on the projecton the project

Completed in 2003Completed in 2003 Continue to work on locating Continue to work on locating

genes and mapping on genes and mapping on specific chromosomesspecific chromosomes

Human Genome ProjectHuman Genome ProjectOutcomes:Outcomes:

Value in knowing the Value in knowing the sequence of genes on sequence of genes on chromosomes, useful in chromosomes, useful in medicine, forensics and medicine, forensics and evolutionevolution

Medical benefits:Medical benefits: Improved diagnosis of Improved diagnosis of

diseasedisease Early detection of genetic Early detection of genetic

susceptibility to diseasesusceptibility to disease Better identification of Better identification of

carriers of genetic carriers of genetic disordersdisorders

Drug design to find new Drug design to find new classes of drugs to act on classes of drugs to act on specific genesspecific genes

Human Genome ProjectHuman Genome ProjectOutcomes:Outcomes:

Improved techniques Improved techniques have made it possible have made it possible to find genomes of to find genomes of other organismsother organisms

Provided new insights Provided new insights into role of “junk DNA” into role of “junk DNA” (sections of DNA that (sections of DNA that are not transcribed)are not transcribed)

Found far fewer Found far fewer protein coding regions protein coding regions than expected than expected

Humans have about Humans have about 25,000 genes25,000 genes

Human Genome ProjectHuman Genome Project

Outcomes cont’dOutcomes cont’d Knowledge of the significance Knowledge of the significance

of certain sequences of certain sequences Presence or absence can be Presence or absence can be

detected using microarray detected using microarray technologytechnology

New research fields have New research fields have emergedemerged

Bioinformatics:Bioinformatics: use of use of computers to store and analyze computers to store and analyze huge amounts of data being huge amounts of data being generated by sequencing the generated by sequencing the genomegenome

Pharmacogenomics:Pharmacogenomics: links links differences in genomic differences in genomic information in different information in different populations to differences in populations to differences in their response to drug their response to drug treatmenttreatment

Cloning Eukaryotic CellsCloning Eukaryotic Cells

1.1. A cell is taken from the desired A cell is taken from the desired organism; an egg cell is taken from a organism; an egg cell is taken from a surrogatesurrogate

2.2. The nucleus is removed from the eggThe nucleus is removed from the egg3.3. The desired cell and the nucleus free The desired cell and the nucleus free

egg are fused togetheregg are fused together4.4. The egg grows to become an embryoThe egg grows to become an embryo5.5. Embryo is implanted in the surrogate Embryo is implanted in the surrogate

and develops into the desired and develops into the desired organismorganism

GMOsGMOs GGenetically enetically MModified odified OOrganisms: rganisms:

organisms in which their genetic make up organisms in which their genetic make up has been alteredhas been altered

Benefits to GM products:Benefits to GM products: Crops:Crops: enhanced taste and quality; reduced enhanced taste and quality; reduced

maturation time; increased nutrients, yields and maturation time; increased nutrients, yields and stress tolerance; improved resistance to stress tolerance; improved resistance to disease, pests and herbicidesdisease, pests and herbicides

Animals:Animals: increased resistance, productivity, increased resistance, productivity, hardiness, and feed efficiency; better yields of hardiness, and feed efficiency; better yields of meat, eggs, and milk; improved animal healthmeat, eggs, and milk; improved animal health

Environment:Environment: “friendly” bioherbicides and “friendly” bioherbicides and bioinsecticides; conservation of soil, water, and bioinsecticides; conservation of soil, water, and energy; better natural waste management; more energy; better natural waste management; more efficient processingefficient processing

Society:Society: increased food security for growing increased food security for growing populationspopulations

GMOsGMOs Controversies:Controversies:

Safety:Safety: potential human health impact (allergies, potential human health impact (allergies, transfer of antibiotic resistance), unknown potential transfer of antibiotic resistance), unknown potential environmental impact, unknown effect on other environmental impact, unknown effect on other organisms, loss of flora and fauna biodiversityorganisms, loss of flora and fauna biodiversity

Access and Intellectual Property:Access and Intellectual Property: domination of domination of world food production by few companies; increased world food production by few companies; increased dependence on industrial nations by developing dependence on industrial nations by developing countries;countries;

Ethics:Ethics: violation of natural organisms’ intrinsic violation of natural organisms’ intrinsic values; tampering with nature by mixing genes values; tampering with nature by mixing genes among species; objections to consuming animal among species; objections to consuming animal genes in plants and vice versa; stress for animalsgenes in plants and vice versa; stress for animals

Labeling:Labeling: not mandated by some countries (US); not mandated by some countries (US); mixing GM crops with non-GM cropsmixing GM crops with non-GM crops

Society:Society: new advances may be skewed to interest new advances may be skewed to interest rich countriesrich countries

GMOsGMOs Current ExamplesCurrent Examples

*Salt tolerance in tomato *Salt tolerance in tomato plant plant

*Synthesis of beta-*Synthesis of beta-carotene in rice (golden carotene in rice (golden rice)rice)

*Factor IX (for human *Factor IX (for human blood clotting) in sheep blood clotting) in sheep milkmilk

Herbicide resistance in Herbicide resistance in crop plants (round-up crop plants (round-up ready plants)ready plants)

Glowing Bacteria… you Glowing Bacteria… you will do this!will do this!

(* = Examples from IB, must know 2)(* = Examples from IB, must know 2)

Issues in BiotechnologyIssues in Biotechnology Benefits and harmful Benefits and harmful

effects of genetic effects of genetic modificationmodification

Ethical Issues of Ethical Issues of therapeutic cloning in therapeutic cloning in humans: creating an humans: creating an embryo to supply embryo to supply embryonic stem cells embryonic stem cells for medical usefor medical use

12-13 need more here12-13 need more here

Cloning Eukaryotic CellsCloning Eukaryotic Cells