Biotechnology & Recombinant DNA

What is biotechnology?

Using living microorganisms or cell components to make products 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 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

This can be used as a vector…

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!

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?

Figure 9.4 (1 of 2)

Stands for _____________

Need primers of about 20 bp to start

DNA polymerase doesn’t start reaction, only lengthensPrimers recognize regions which flank target gene

IngredientsprimersDNA polymerase from TAC (Thermus aquaticus) Individual nucleotides

Can you go over that one more time? …PCR animation

What is PCR?

Figure 9.4 (2 of 2)

Stands for _____________

Need primers of about 20 bp to start

DNA polymerase doesn’t start reaction, only lengthensPrimers recognize regions which flank target gene

IngredientsprimersDNA polymerase from TAC (Thermus aquaticus) Individual nucleotides

Can you go over that one more time? …PCR animation

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

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)

Gel electrophoresis animation

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

DNA fingerprinting for identification

Figure 9.17

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

Transformation Electroporation Protoplast fusion Microinjection Gene gun

What is RNA Interference (RNAi)?

Figure 9.14

RNAi animation

Cloning

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… Must make

complementary DNA (cDNA)

Use reverse trasncriptase

cDNA animation Now DNA can be

inserted…

What do they do with the cloned DNA?

Lots of different things!

Is this the only way to “look” at DNA? No—we can sequence it, too! 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 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

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

What is gene therapy? 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