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Ch. 20 Biotechnology Objective: LO 3.5 The student can justify the claim that humans can manipulate heritable information by identifying at least two commonly used technologies.
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Ch. 20 Biotechnology Objective: LO 3.5 The student can justify the claim that humans can manipulate heritable information by identifying at least two commonly.

Apr 01, 2015

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Page 1: Ch. 20 Biotechnology Objective: LO 3.5 The student can justify the claim that humans can manipulate heritable information by identifying at least two commonly.

Ch. 20 Biotechnology

Objective:LO 3.5 The student can justify the claim that humans can manipulate heritable information by identifying

at least two commonly used technologies.

Page 2: Ch. 20 Biotechnology Objective: LO 3.5 The student can justify the claim that humans can manipulate heritable information by identifying at least two commonly.

Understanding and Manipulating Genomes

• Sequenced the human genome in 2003 through:– Biotechnology: manipulation of organisms

• Genetic engineering: manipulation of genes– Recombinant DNA: 2 DNAs combined

Page 3: Ch. 20 Biotechnology Objective: LO 3.5 The student can justify the claim that humans can manipulate heritable information by identifying at least two commonly.

Using Bacteria as Tools

• Bacteria– Circular DNA– Plasmid

• Extra genetic material• Small, circular DNA• Not necessary, but usually

beneficial

http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/R/RecombinantPlasmid.gif

Page 4: Ch. 20 Biotechnology Objective: LO 3.5 The student can justify the claim that humans can manipulate heritable information by identifying at least two commonly.

Using Bacteria as Tools

• Bacterial Transformation– Uptake of DNA from the

fluid surrounding the cell– Causes genetic

recombination– Allow insertion of gene

of interest

http://biology200.gsu.edu/houghton/4564%20'04/figures/lecture%203/transformation.jpg

Page 5: Ch. 20 Biotechnology Objective: LO 3.5 The student can justify the claim that humans can manipulate heritable information by identifying at least two commonly.

20.1: DNA (Gene) Cloning• Uses: make many

copies (amplify) quickly and produces proteins

• Basic Method:1. Use bacterial plasmids

(cloning vector).2. Insert desired gene

(recombinant DNA).3. Return plasmid to

bacteria.4. Bacteria reproduce.5. Various applications.

Page 6: Ch. 20 Biotechnology Objective: LO 3.5 The student can justify the claim that humans can manipulate heritable information by identifying at least two commonly.

Making Recombinant DNA

• Restriction enzymes (nucleases) cut DNA in specific places (restriction site) to form restriction fragments.– Must use same enzyme on

plasmid and desired gene– Forms sticky ends:

unbonded nucleotides– Add DNA ligase to rebond

recombinant DNA.

Page 7: Ch. 20 Biotechnology Objective: LO 3.5 The student can justify the claim that humans can manipulate heritable information by identifying at least two commonly.

Cloning a Eukaryotic Gene in a Bacterial Plasmid

• In gene cloning, the original plasmid is called a cloning vector

• A cloning vector is a DNA molecule that can carry foreign DNA into a cell and replicate there

Page 8: Ch. 20 Biotechnology Objective: LO 3.5 The student can justify the claim that humans can manipulate heritable information by identifying at least two commonly.

Cloning a Eukaryotic Gene in a Bacterial Plasmid

• Only a cell that took up a plasmid, which has the ampR gene, will reproduce and form a colony.

– Colonies with nonrecombinant plasmids will be blue, because they can hydrolyze X-gal.

– Colonies with recombinant plasmids, in which lacZ is disrupted, will be white, because they cannot hydrolyze X-gal.

• By screening the white colonies with a nucleic acid probe (see Figure 20.5), researchers can identify clones of bacterial cells carrying the gene of interest.

Isolate plasmid DNAand human DNA.

Cut both DNA samples withthe same restriction enzyme.

Mix the DNAs; they join by base pairing.The products are recombinant plasmidsand many nonrecombinant plasmids.

Bacterial cell lacZ gene(lactosebreakdown)

Humancell

Restrictionsite

ampR gene(ampicillinresistance)

Bacterialplasmid Gene of

interest

Stickyends

Human DNAfragments

Recombinant DNA plasmids

Introduce the DNA into bacterial cellsthat have a mutation in their own lacZgene.

Recombinantbacteria

Plate the bacteria on agarcontaining ampicillin and X-gal.Incubate until colonies grow.

Colony carrying non-recombinant plasmidwith intact lacZ gene

Colony carryingrecombinantplasmid withdisrupted lacZ gene

Bacterialclone

Isolate plasmid DNAand human DNA.

Cut both DNA samples withthe same restriction enzyme.

Mix the DNAs; they join by base pairing.The products are recombinant plasmidsand many nonrecombinant plasmids.

Bacterial cell lacZ gene(lactosebreakdown)

Humancell

Restrictionsite

ampR gene(ampicillinresistance)

Bacterialplasmid Gene of

interest

Stickyends

Human DNAfragments

Recombinant DNA plasmids

Introduce the DNA into bacterial cellsthat have a mutation in their own lacZgene.

Recombinantbacteria

Plate the bacteria on agarcontaining ampicillin and X-gal.Incubate until colonies grow.

Colony carrying non-recombinant plasmidwith intact lacZ gene

Colony carryingrecombinantplasmid withdisrupted lacZ gene

Bacterialclone

Page 9: Ch. 20 Biotechnology Objective: LO 3.5 The student can justify the claim that humans can manipulate heritable information by identifying at least two commonly.

Storing Cloned Genes• Genomic Library: complete set of plasmid clones saved.• Phages are also used so they are saved as phage library.• A bacterial artificial chromosome (BAC) is a large plasmid

that has been trimmed down and can carry a large DNA insert• Complementary DNA (cDNA) can be made by reverse

transcription of mRNA to make a cDNA library.

Page 10: Ch. 20 Biotechnology Objective: LO 3.5 The student can justify the claim that humans can manipulate heritable information by identifying at least two commonly.

ID Clone Carrying Gene of Interest• Nucleic acid probe (RNA or DNA) radioactively

labeled which hybridizes to gene.

Master plate

Filter

Solutioncontainingprobe

Filter liftedand flipped over

Radioactivesingle-strandedDNA

ProbeDNA

Gene ofinterest

Single-strandedDNA from cell

Film

Hybridizationon filter

Master plate

Coloniescontaininggene ofinterest

A special filter paper is pressed against the master plate, transferring cells to the bottom side of the filter.

The filter is treated to break open the cells and denature their DNA; the resulting single-stranded DNA molecules are treated so that they stick to the filter.

The filter is laid under photographic film, allowing any radioactive areas to expose the film (autoradiography).

After the developed film is flipped over, the reference marks on the film and master plate are aligned to locate colonies carrying the gene of interest.

Master plate

Filter

Solutioncontainingprobe

Filter liftedand flipped over

Radioactivesingle-strandedDNA

ProbeDNA

Gene ofinterest

Single-strandedDNA from cell

Film

Hybridizationon filter

Master plate

Coloniescontaininggene ofinterest

A special filter paper is pressed against the master plate, transferring cells to the bottom side of the filter.

The filter is treated to break open the cells and denature their DNA; the resulting single-stranded DNA molecules are treated so that they stick to the filter.

The filter is laid under photographic film, allowing any radioactive areas to expose the film (autoradiography).

After the developed film is flipped over, the reference marks on the film and master plate are aligned to locate colonies carrying the gene of interest.

Page 11: Ch. 20 Biotechnology Objective: LO 3.5 The student can justify the claim that humans can manipulate heritable information by identifying at least two commonly.

Eukaryotic Genes in Bacterial Expression Systems

• To overcome differences in promoters and other DNA control sequences, scientists usually employ an expression vector, a cloning vector that contains a highly active prokaryotic promoter

• To overcome inability to remove introns, use cDNA form of the gene

Page 12: Ch. 20 Biotechnology Objective: LO 3.5 The student can justify the claim that humans can manipulate heritable information by identifying at least two commonly.

Eukaryotic Cloning and Expression Systems

• The use of cultured eukaryotic cells as host cells and yeast artificial chromosomes (YACs) as vectors helps avoid gene expression problems

• YACs behave normally in mitosis and can carry more DNA than a plasmid

• Eukaryotic hosts can provide the posttranslational modifications that many proteins require http://www.accessexcellence.org/RC/VL/GG/images/YAC.gif

Page 13: Ch. 20 Biotechnology Objective: LO 3.5 The student can justify the claim that humans can manipulate heritable information by identifying at least two commonly.

Amplifying DNA: Polymerase Chain Reaction

1 DNA strand → billions in hours.1. Denature: Heat DNA to break

H-bonds2. Annealing: Add primers and

cool3. Extension: Add heat resistant

DNA polymerase and nucleotides

4. Repeat using thermocycler

Genomic DNA

Targetsequence

5

3

3

5

5

3

3

5

Primers

Denaturation:Heat brieflyto separate DNAstrands

Annealing:Cool to allowprimers to formhydrogen bondswith ends oftarget sequence

Extension:DNA polymeraseadds nucleotides tothe 3 end of eachprimer

Cycle 1yields

2molecules

Newnucleo-

tides

Cycle 2yields

4molecules

Cycle 3yields 8

molecules;2 molecules

(in white boxes)match target

sequence

Genomic DNA

Targetsequence

5

3

3

5

5

3

3

5

Primers

Denaturation:Heat brieflyto separate DNAstrands

Annealing:Cool to allowprimers to formhydrogen bondswith ends oftarget sequence

Extension:DNA polymeraseadds nucleotides tothe 3 end of eachprimer

Cycle 1yields

2molecules

Newnucleo-

tides

Cycle 2yields

4molecules

Cycle 3yields 8

molecules;2 molecules

(in white boxes)match target

sequence

Page 14: Ch. 20 Biotechnology Objective: LO 3.5 The student can justify the claim that humans can manipulate heritable information by identifying at least two commonly.

20.2 Restriction Fragment AnalysisGel Electrophoresis• DNA is – charge; attracted

to +• Gel that separates DNA by

length; smaller pieces can travel faster/further.

• Make fragments by restriction enzymes and separate them.– Alleles have different

sequences of DNA so are cut differently.

Normal -globin allele

175 bp 201 bp Large fragment

Sickle-cell mutant -globin allele

376 bp Large fragment

Ddel Ddel Ddel Ddel

Ddel Ddel Ddel

Ddel restriction sites in normal and sickle-cell alleles of-globin gene

Normalallele

Sickle-cellallele

Largefragment

376 bp201 bp175 bp

Electrophoresis of restriction fragments from normaland sickle-cell alleles

Page 15: Ch. 20 Biotechnology Objective: LO 3.5 The student can justify the claim that humans can manipulate heritable information by identifying at least two commonly.

Southern Blotting• A technique called

Southern blotting combines gel electrophoresis with nucleic acid hybridization

• Specific DNA fragments can be identified by Southern blotting, using labeled probes that hybridize to the DNA immobilized on a “blot” of gel

DNA + restriction enzyme Restrictionfragments

Normal-globinallele

Sickle-cellallele

Heterozygote

Preparation of restriction fragments. Gel electrophoresis. Blotting.

Nitrocellulosepaper (blot)

Gel

Sponge

Alkalinesolution

Papertowels

Heavyweight

Hybridization with radioactive probe.

Radioactivelylabeled probefor -globingene is addedto solution ina plastic bag

Paper blot

Probe hydrogen-bonds to fragmentscontaining normalor mutant -globin

Fragment fromsickle-cell-globin allele

Fragment fromnormal -globinallele

Autoradiography.

Film overpaper blot

DNA + restriction enzyme Restrictionfragments

Normal-globinallele

Sickle-cellallele

Heterozygote

Preparation of restriction fragments. Gel electrophoresis. Blotting.

Nitrocellulosepaper (blot)

Gel

Sponge

Alkalinesolution

Papertowels

Heavyweight

Hybridization with radioactive probe.

Radioactivelylabeled probefor -globingene is addedto solution ina plastic bag

Paper blot

Probe hydrogen-bonds to fragmentscontaining normalor mutant -globin

Fragment fromsickle-cell-globin allele

Fragment fromnormal -globinallele

Autoradiography.

Film overpaper blot

Page 16: Ch. 20 Biotechnology Objective: LO 3.5 The student can justify the claim that humans can manipulate heritable information by identifying at least two commonly.

Restriction Fragment Length Polymorphisms (RFLPs)

• Restriction fragments made using the same enzyme on homologues.

• Used as a marker (fingerprint) for individuals.

Paternity Test

Page 17: Ch. 20 Biotechnology Objective: LO 3.5 The student can justify the claim that humans can manipulate heritable information by identifying at least two commonly.

DNA Sequencing

• Relatively short DNA fragments can be sequenced by the dideoxy chain-termination method

• Inclusion of special dideoxyribonucleotides in the reaction mix ensures that fragments of various lengths will be synthesized

Page 18: Ch. 20 Biotechnology Objective: LO 3.5 The student can justify the claim that humans can manipulate heritable information by identifying at least two commonly.

DNA SequencingDNA(template strand)

5

3

Primer3

5

DNApolymerase

Deoxyribonucleotides Dideoxyribonucleotides(fluorescently tagged)

3

5DNA (templatestrand)

Labeled strands3

Directionof movementof strands

Laser Detector

Page 19: Ch. 20 Biotechnology Objective: LO 3.5 The student can justify the claim that humans can manipulate heritable information by identifying at least two commonly.

DNA Sequencing

http://files.myweb.med.ucalgary.ca/files/64/images/DNA%20Sequencing%20Images/Sample_sequencing_result_2005-10-25_copy.jpg

Page 20: Ch. 20 Biotechnology Objective: LO 3.5 The student can justify the claim that humans can manipulate heritable information by identifying at least two commonly.

How to ID Unknown Genes

• Compare to known genes of other organisms.• Disable the gene and observe the

consequence.– In vitro interference: use copies DNA gene,

introduce mutagen, reinsert into cell, observe consequence.

Page 21: Ch. 20 Biotechnology Objective: LO 3.5 The student can justify the claim that humans can manipulate heritable information by identifying at least two commonly.

Studying Expression of Interacting Groups of Genes

• DNA Microarray Assays– Take mRNA– Make cDNA (single

strand)– Fluorescently label– Apply to array chip

(contains known DNA fragments the cDNA will bond to)

– Look for fluorescence.

Make cDNA by reverse transcription, using fluorescently labeled nucleotides.

Apply the cDNA mixture to a microarray, a microscope slide on which copies of single-stranded DNA fragments from the organism’s genes are fixed, a different gene in each spot. The cDNA hybridizes with any complementary DNA on the microarray.

Rinse off excess cDNA; scan microarray for fluorescent. Each fluorescent spot (yellow) represents a gene expressed in the tissue sample.

Isolate mRNA.Tissue sample

mRNA molecules

Labeled cDNA molecules(single strands)

DNAmicroarray

Size of an actualDNA microarraywith all the genesof yeast (6,400 spots)

Page 22: Ch. 20 Biotechnology Objective: LO 3.5 The student can justify the claim that humans can manipulate heritable information by identifying at least two commonly.

Determining Gene Function• One way to determine function is to disable the gene and

observe the consequences (knock-outs)• Using in vitro mutagenesis, mutations are introduced into a

cloned gene, altering or destroying its function• When the mutated gene is returned to the cell, the normal

gene’s function might be determined by examining the mutant’s phenotype

A transgenic mouse with an active rat growth hormone gene (left). This transgenic mouse is twice the size of a normal mouse (right). http://web.virginia.edu/Heidi/chapter29/Images/8883n29_30.jpg

Page 23: Ch. 20 Biotechnology Objective: LO 3.5 The student can justify the claim that humans can manipulate heritable information by identifying at least two commonly.

Comparing Genomes of Different Species

• Allows us to look for evolutionary relationships.• Comparative data on simple organisms helps us

understand more complex ones.• Closely related species: figure out one and use as a

template for the others.

Page 24: Ch. 20 Biotechnology Objective: LO 3.5 The student can justify the claim that humans can manipulate heritable information by identifying at least two commonly.

Future Directions

• Proteomics: study proteins encoded by genomes.

• Single Nucleotide Polymorphisms (SNPs): single base-pair differences from one human to another.– People are 99.99% identical on genetic level.

Page 25: Ch. 20 Biotechnology Objective: LO 3.5 The student can justify the claim that humans can manipulate heritable information by identifying at least two commonly.

20.3 Cloning• In Plants:

– Totipotent: cells can dedifferentiate.– Tranplanting a clipping or root causing a clone to

be made.

Page 26: Ch. 20 Biotechnology Objective: LO 3.5 The student can justify the claim that humans can manipulate heritable information by identifying at least two commonly.

Cloning

• In Animals– Remove nucleus from

egg– Add nucleus from

somatic cell of donor– Grow in culture– Implant in uterus– Clone is born!

Page 27: Ch. 20 Biotechnology Objective: LO 3.5 The student can justify the claim that humans can manipulate heritable information by identifying at least two commonly.

CC, the first cloned cat

Although CC is a clone of her mother, they are not identical due to the X-inactivation mechanism and different environmental influences Figure 20.20

Page 28: Ch. 20 Biotechnology Objective: LO 3.5 The student can justify the claim that humans can manipulate heritable information by identifying at least two commonly.

Stem Cells of Animals

• Goal of cloning human embryos → stem cell production

• Stem cell = undifferentiated cell

• Embryonic stem cells have the potential to become anything (pluripotent).

• Adult stem cells can’t.

Page 29: Ch. 20 Biotechnology Objective: LO 3.5 The student can justify the claim that humans can manipulate heritable information by identifying at least two commonly.

Regenerative Medicine?

• Human pluripotent stem cells crucial for the development of regenerative medicine

• Can allow for growing a whole new heart or liver, since they can be converted into any cell type in the body

Human ear grown in a lab from stem cells.

http://www.zmescience.com/research/studies/lab-grown-stem-cells-may-mutate-in-time/

Page 30: Ch. 20 Biotechnology Objective: LO 3.5 The student can justify the claim that humans can manipulate heritable information by identifying at least two commonly.

20.4 Applications of Genetic Engineering

• Medical Applications:– Identifying genes that

cause disease/disordersNormal -globin allele

175 bp 201 bp Large fragment

Sickle-cell mutant -globin allele

376 bp Large fragment

Ddel Ddel Ddel Ddel

Ddel Ddel Ddel

Ddel restriction sites in normal and sickle-cell alleles of-globin gene

Normalallele

Sickle-cellallele

Largefragment

376 bp201 bp175 bp

Electrophoresis of restriction fragments from normaland sickle-cell alleles

– Gene therapy: changing disease causing genes in humans.

Cloned gene

Retroviruscapsid

Bonemarrowcell frompatient

Inject engineeredcells into patient.

Insert RNA version of normal alleleinto retrovirus.

Viral RNA

Let retrovirus infect bone marrow cellsthat have been removed from thepatient and cultured.

Viral DNA carrying the normalallele inserts into chromosome.

Bonemarrow

Page 31: Ch. 20 Biotechnology Objective: LO 3.5 The student can justify the claim that humans can manipulate heritable information by identifying at least two commonly.

20.4 Applications of Genetic Engineering

• Pharmaceutical Products– Insulin– Human growth hormone– Tissue plasminogen

activator to dissolve blood clots

– HIV blockers– Vaccines

http://www.udel.edu/physics/scen103/CGZ/14b.gif

Page 32: Ch. 20 Biotechnology Objective: LO 3.5 The student can justify the claim that humans can manipulate heritable information by identifying at least two commonly.

• Forensic Evidence– DNA fingerprinting

using gel electrophoresis

• Environmental Cleanup– Mining bacteria (copper,

lead, nickel, etc)– Cleaning toxic waster – Clean oil spills

Page 33: Ch. 20 Biotechnology Objective: LO 3.5 The student can justify the claim that humans can manipulate heritable information by identifying at least two commonly.

• Agricultural Applications– Animal Husbandry and

“Pharm” animals• Transgenic animals (has

recombinant DNA) to make better wool, leaner meat, shorter maturation time, pharmaceutical factories for blood clotting factors.

– Genetic Engineering in Plants

• Delayed ripening, resistance to spoilage/disease, increase nutritional value.

• Uses Ti plasmid recombined with desired genes.

Page 34: Ch. 20 Biotechnology Objective: LO 3.5 The student can justify the claim that humans can manipulate heritable information by identifying at least two commonly.

Transgenic Animals

• Human gene for antithrombin inserted into a goat’s genome and the protein is produced in the milk

http://www.livinghistoryfarm.org/farminginthe70s/crops_12.html

Page 35: Ch. 20 Biotechnology Objective: LO 3.5 The student can justify the claim that humans can manipulate heritable information by identifying at least two commonly.

Genetic Engineering in Plants

• Agricultural scientists have endowed a number of crop plants with genes for desirable traits

• The Ti plasmid is the most commonly used vector for introducing new genes into plant cells

Agrobacterium tumefaciens

Tiplasmid

Site whererestrictionenzyme cuts

DNA withthe geneof interest

T DNA

RecombinantTi plasmid

Plant withnew trait

Page 36: Ch. 20 Biotechnology Objective: LO 3.5 The student can justify the claim that humans can manipulate heritable information by identifying at least two commonly.

Transgenic Plants

1994. Flavr Savr Tomato. 1st engineered food in stores. Engineered to remain firm even as it turns red and ripe.

Bt transgenic corn is normal corn that contains a gene from the soil bacterium Bacillus thuringiensis. Gene allows production of a toxic protein that can kill many types of caterpillars(http://www.ces.ncsu.edu/plymouth/pubs/btcorn99.html)

Page 37: Ch. 20 Biotechnology Objective: LO 3.5 The student can justify the claim that humans can manipulate heritable information by identifying at least two commonly.

• Safety and Ethics– Potential benefits of genetic engineering must be

weighed against potential hazards of creating harmful products or procedures

– Most public concern about possible hazards centers on genetically modified (GM) organisms used as food