Download - Ch. 20 Biotechnology
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.
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
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
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
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.
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.
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
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
interestStickyends Human DNA
fragments
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
interestStickyends Human DNA
fragments
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
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.
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.
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
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
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
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 DdelDdel 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
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
Restriction Fragment Length Polymorphisms (RFLPs)
• Restriction fragments made using the same enzyme on homologues.
• Used as a marker (fingerprint) for individuals.
Paternity Test
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
DNA SequencingDNA(template strand)5
3
Primer3
5
DNApolymerase
Deoxyribonucleotides Dideoxyribonucleotides(fluorescently tagged)
3
5DNA (templatestrand)
Labeled strands 3
Directionof movementof strands
Laser Detector
DNA Sequencing
http://files.myweb.med.ucalgary.ca/files/64/images/DNA%20Sequencing%20Images/Sample_sequencing_result_2005-10-25_copy.jpg
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.
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)
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
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.
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.
20.3 Cloning• In Plants:
– Totipotent: cells can dedifferentiate.– Tranplanting a clipping or root causing a clone to
be made.
Cloning
• In Animals– Remove nucleus from
egg– Add nucleus from
somatic cell of donor– Grow in culture– Implant in uterus– Clone is born!
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
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.
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/
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 DdelDdel 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
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
• Forensic Evidence– DNA fingerprinting
using gel electrophoresis
• Environmental Cleanup– Mining bacteria (copper,
lead, nickel, etc)– Cleaning toxic waster – Clean oil spills
• 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.
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
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
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)
• 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