Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings p.174
Biotechnology - use of living organisms to create products or help processes
Ex. HGH, insulin
Recombinant DNA - segment of DNA containing sequences from different organisms
How is DNA manipulated?
A A
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Restriction enzymes cut DNA at specific sites and create sticky ends
Complementary ends will fuse to produce a long strand of DNA
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• The DNA is then integrated into the recipient cell’s chromosome
Figure 12.1D
Donated DNA
Recipient cell’schromosome
Crossovers
Degraded DNA
Recombinantchromosome
bacterium
bacterialchromosome
plasmid
Plasmids are extra rings of DNA that replicate in bacteria.DNA can be inserted into plasmids.
Cloning Vectors
1. Use restriction enzymes.
2. Insert gene into plasmid.
3. Transfer the plasmid backinto bacterial cell.
4. Let bacterial cells replicate.
bacterialclones
replication
transformation
recombinant DNA
Plasmid
BacteriumHuman cell
DNA
Human proteinBacterialchromosome
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Recombinant DNA products
• “seed” protein for artificial snow
• Insulin for diabetes treatment
• Enzymes that clean up toxic waste spills
• Growth Hormones (Human, Bovine)
• TPA: Tissue Plasminogen Activator for treatment of heart attacks
Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings
• The polymerase chain reaction (PCR) can quickly clone a small sample of DNA in a test tube
• Selection of specific sequence
Figure 12.12
InitialDNAsegment
1 2 4 8
Number of DNA molecules
Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings
Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings
• Restriction fragments of DNA are compared by size
Gel electrophoresis sorts DNA molecules by size
Figure 12.10
Mixture of DNAmolecules ofdifferent sizes
Powersource
Gel
Glassplates
Longermolecules
Shortermolecules
Completed gel
DNA forensics
Egg manipulation via microinjection.
Credit: © Science VU/Visuals Unlimited
Egg microinjection to produce transgenic animal
Grow bigger fish faster. Salmon with gene from another fish species
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• Uses of transformed animals:
• Produce medicines more easily
Ex. sheep and gene to treat cystic fibrosis
Goats and AT3 gene to prevent blood clots
Figure 12.16
Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings Fig. 11-14, p.173
An extremely large Agrobacterium tumefaciens tumor (crown gall disease) and secondary tumors on Kalanchoe stem.
Credit: © Brad Mogen
Genetic engineering of plants
Methods to insert DNA:
1. Ballistics2. Protoplasts3. Agrobacterium as vector
- Ti plasmid
a A bacterial cell contains a Ti plasmid (purple) that has a foreign gene (blue).
b The bacterium infects a plant and transfers the Ti plasmid into it. The plasmid DNA becomes integrated into one of the plant’s chromosomes.
c The plant cell divides. Its descendant cells form an embryo, which may develop into a mature plant that can express the foreign gene.
A young plant
expressing a fluorescent gene product
Fig. 11-12, p.171
Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings
Genetically modified crops
• Golden rice with Vitamin A
• Cotton resistant to boll weevil
• Soybeans resistant to herbicide (Roundup)
• Corn resistant to European corn borer
• Rapeseed with healthier vegetable oil
Benefits and risks
white sheep
black sheep
egg cell
udder cells
DNA
embryo
Dollysurrogatemother
How Dolly was cloned
Cloning of human cells
• Regenerative medicine– Bone, pancreas cells, skin
Stem cells - the $6 billion promise?
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• treat disease by altering an afflicted individual’s genes
– Ex vivo
– In vivo
– Stem cells
Gene therapy may someday help treat a variety of diseases
Figure 12.19
Cloned gene (normal allele)
1 Insertnormal geneinto virus
Viral nucleicacid
Retrovirus
2 Infect bonemarrow cellwith virus
3 Viral DNAinserts intochromosome
Bone marrowcell from patient
Bone marrow
4 Inject cellsinto patient
Human Genome Project
• 3.2 billion bases in 22 autosomes + X, Y
• Draft sequence completed in 2003
• Available at www.ornl.gov/sci/techresources/Human_Genome/home.shtml
• www.ucsc.genome.edu
What does the human genome sequence tell us?• 20 K to 25 K genes• 99.9% alike, across all races• 97% of DNA is not transcribed
- Spacers between genes- Structural (centromeres, telomeres)
- Regulatory (enhancers, promoters)
- Leftovers of evolution?
Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings
How are specific genes identified?
1. Isolate it from a genomic library by homology with a gene from another organism.
2.Find mRNA for the gene, make cDNA from it.
3.Make DNA sequence based on protein sequence.
Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings
1. Nucleic acid probes identify clones carrying specific genes
Figure 12.8A
Radioactiveprobe (DNA)
Mix with single-stranded DNA fromvarious bacterial(or phage) clones
Single-strandedDNA
Base pairingindicates thegene of interest
• A nucleic acid probe can tag a desired gene in a library
Fig. 11-4, p.164
mRNA
mRNA
cDNA
DNA
DNA
reversetranscriptase
DNApolymerase
cDNA
• Complementary DNA
• Using reverse transcriptase
• Assembles DNA on mRNA template
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• A labeled probe can reveal patterns of gene expression in different kinds of cells
DNA microarrays test for the expression of many genes at once
Figure 12.9
cDNA
DNA of gene
DNAmicroarray,actual size(6,400 genes)
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Gene Therapy
• What is it?
• How is it done?
• Does it work?
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Gene therapy
• Goal - Treat diseases caused by mutated genes
• Method - Add a normal gene or block an abnormal gene in enough cells to restore normal function
• Target - somatic cells
Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings
Which disorders are candidates for gene therapy treatment?
• Disorders due to mutations in one or more genes
• The responsible gene is known
• The affected tissues are known and accessible
Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings
Knockout gene therapy
• Goal: turn off a gene that is causing a disorder
• Strategies:
– Antisense
– Triple helix oligos
– Spliceosome
– Ribozyme
Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings
How is gene therapy done?
1. Identify the gene(s) responsible for the disorder
2. Make copies of the normal gene
3. Insert the copies into vectors (i.e., viruses)
4. Infect the affected cells with the vectors
5. Activate the gene
– Transcription and translation take place
Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings
Critical factors in choosing a vector
Gene size
– Limited room in vector genome
• Target tissue
– What cells can the vector infect?
• Integration into the genome
– Without integration, only short-term effect
– Random integration may disrupt other genes
• Cell cycle stage (dividing vs. non-dividing)
Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings
Gene Therapy Successes
Ashanti de Silva successfully treated for ADA deficiency - 1990
Ryes Evans successfully treated for SCID - 2001
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ilva
Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings
Gene Therapy Problems
Jesse Gelsinger died of complications due to an immune system response while participating in a clinical trial
Three children treated for SCID developed leukemia due to disruption of a gene that regulates cell division
Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings
Ethical and Social Issues
• Patient safety while participating in clinical trials
• Which applications are therapies and which are enhancements?
– “Designer” babies
• Access to gene therapies