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Biotechnology & Recombinant DNA
What is biotechnology?
Using living microorganisms or cell components to make products
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Often via genetic engineering and using recombinant DNA
Recombinant DNA technology:
Insertion or modification of genes to produce desired proteins.
What is recombinant DNA?
Inserting foreign DNA into a bacterial cell
Use restriction enzymes, plasmids, ligase and bacterial host
Restriction enzymes
 Defense against viruses
Clone gene or make gene product
How is recombinant DNA made?
Recall: happens naturally
 Transposons
Researchers can also produce it
 Use bacterial plasmids or viruses (bacteriophages)
Uses
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Bacteria make insulin
yeast help make components of Hepatitis B vaccine
What are restriction enzymes?
Naturally occur in bacteria
 Bacteria use these to combat viral infection
 Bacteria DNA uneffected because some is methylated
Sequence-specific
Restriction enzymes
Cloning animation
What is a vector?
Different from a disease vector!
plasmid animation
Plasmid or virus
 Used to insert DNA into host cell
 Must be able to self-replicate!
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Must be small so not fragile
Both types of vectors can allow researchers to clone DNA
 But there’s another approach to DNA amplification…
PCR: making copies of DNA
What is PCR?
What is PCR?
Why would you use PCR?
Detect small amounts of DNA
 Can you think of examples?
 Get in groups and discuss!
Forensics
Infectious agents
Gene mapping
 Human Genome Project
Taxonomy and systematics studies
Cancer and study of other human diseases
Sequencing of rRNA and mRNA via cDNA
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DNA fingerprinting
Let’s take a brief look at some of these…
What is gel electrophoresis?
Sorting DNA segments by size
DNA fingerprinting
 Restriction enzymes create restriction fragment length polymorphisms (RFLPs)
What is Southern blotting?
After gel electrophoresis
Filter paper blots DNA off
Radioactive probes added
Autoradiography pinpoints sequence
Southern Blot animation
The scientific applications
Understanding of DNA
Sequencing organisms' genomes
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DNA fingerprinting for identification
Why E. Coli is used!
Used because it is easily grown and its genomics are known
Need to eliminate endotoxin from products
Cells must be lysed to get product
Inserting Foreign DNA into Cells
DNA can be inserted into a cell by
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Transformation
Electroporation
Protoplast fusion
Microinjection
Gene gun
What is RNA Interference (RNAi)?
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How do researchers get the DNA they want to clone?
Gene libraries are made of pieces of an entire genome stored in plasmids or phages.
cDNA is made from mRNA by reverse transcriptase.
Synthetic DNA is made by a DNA synthesis machine.
So what’s the problem?
Fine for prokaryotic DNA
Problem with eukaryotic DNA…
Eukaryotic DNA has introns…
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Must make complementary DNA (cDNA)
Use reverse trasncriptase
cDNA animation
Now DNA can be inserted…
What do they do with the cloned DNA?
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Lots of different things!
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Is this the only way to “look” at DNA?
No—we can sequence it, too!
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Cloning
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Random shotgun sequencing
Start with a whole genome or a large piece of the DNA (a BAC).
 BAC-bacterial artificial choromosme
Shear the DNA into many different, random segments.
Sequence each of the random segments Put the pieces back together in original order
What are bioreactors?
Using bacteria to produce gene products
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Insulin: diabetes
Human growth hormone
Cellulase (break down cell wall for animal feed)
Factor VIII: hemophilia
What are plant GMOs?
Genetically modified organisms
 Transgenic plant or animal
Bioreactors filled with these
Plants
 Cotton, corn, potato to make pest resistant
 Soybeans resistant to common herbicide
 Some corn, cotton are herbicide and pest resistant
 Could produce human hormones, clotting factors, antibodies on seeds in future
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What are animal GMOs?
Foreign genes into embryos
produce animals that manufacture human hormones, etc. = gene pharming
 Blood clotting factor goats
 Sheep milk with human alpha-1-antitrypsin (used to treat heritable emphysema)
1997, Dolly
Since then, cloned sheep, cows, goats, mice
Humans: moratorium
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What is gene therapy?
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Insertion of genetic material into human cells to treat a disorder
 Use retrovirus to insert normal gene into cell
Healthy genes to make up for faulty genes
 Severe combined immunodeficiency syndrome
 1990, girl received normal gene in white blood cells
Using genes to treat other illnesses