Chapter 8

Biotechnology and Recombinant DNACH 8 MMB due Sunday night!

Biotechnology and Recombinant DNA

• ______________: the use of microorganisms, cells, or cell components to make a product– Foods, antibiotics, vitamins, enzymes

• ___________________(rDNA) technology: insertion or modification of genes to produce desired proteins

Biotechnology and Recombinant DNA

• ___________: self-replicating DNA used to carry the desired gene to a new cell– Plasmids and viruses can be used as vectors

A Typical Genetic Modification Procedure.

bacterium

recombinant DNA(plasmid)

transformed bacterium

Plasmid

Bacterial cromosome

Vector, such as a plasmid, is isolated.

DNA containing gene of interest from a different species is cleaved by an enzyme into fragments.

Desired gene is selected and inserted into plasmid.

Plasmid is taken up by a cell, such as a bacterium.

Cells with gene of interest are cloned with either of two goals in mind.Create and harvest

copies of a gene. Create and harvest protein products of a gene.

PlasmidRNAProtein product

or

Gene encoding protein for pest resistance is inserted into plant cells.

Gene encoding degradative enzyme to clean up toxic waste is inserted into bacterial cells.

Amylase, cellulase, and other enzymes prepare fabrics for clothing manufacture.

Human growth hormone treats stunted growth.

DNA containing gene of interest

Extracting DNA

• A source of DNA is needed for manipulation in Biotechnology applications

•

• Performed by disrupting the cell wall and/or membrane by chemical or mechanical means and then collect the cell free DNA

Polymerase chain reaction

•

• Used to:– Amplify DNA to detectable levels– Sequence DNA– Detect pathogens

PCR• Target region can be for any gene of interest,

usually short portions of the gene less than 1000 bp’s (base pairs)

• Very fast 2-3 hours• Primers are used to selectively replicate only

the target region– Short sections of DNA that base pair to single

stranded DNA–

PCR

• PCR is possible due to ___________________– _________________________from a thermophile

Thermus aquaticus– Stable at temperatures up to 95 degrees C– DNA must be heated to ~95 C which breaks

hydrogen bonds of DNA • Double stranded single stranded

Incubate target DNA at 94°C for 1 minute to separate the strands.

Add primers, nucleotides (deoxynucleotides, dNTP), and DNA polymerase.

Primers attach to single-stranded DNA during incubation at 60°C for 1 minute.

Incubate at 72°C for 1 minute; DNA polymerase copies the target DNA at this temperature.

Repeat the cycle of heating and cooling to make two more copies of target DNA.

Target DNA

Primer

DNA polymerase

dNTP

Firs

t cyc

leSe

cond

cyc

leThe polymerase chain reaction.

© 2013 Pearson Education, Inc.

Medical Applications• DNA extractions and PCR is often used in the medical

field to confirm diagnosis or to identify organisms causing disease.

• Bordatella pertussis or causative agent for whooping cough. – aerobic Gram negative bacillus – B. pertussis is very difficult to isolate due to its extreme

sensitivity to desiccation• DNA is first extracted from specimen and specific primers

targeting regions specific to B. pertussis are used in PCR. • The presence of the genes specific for B. pertussis

confirm that the patient does indeed have pertussis. • http://

www.whoopingcough.net/cough-child-muchwhooping.wav

• – Allow scientist to __________________________– Each enzyme recognizes a 4-6 base pair nucleotide

sequence

PCR Applications

Selected Restriction Enzymes Used in rDNA Technology

Sticky endsFor recombinant DNA techniques

Blunt ends

DNA Fingerprinting

• Uniquely identifies individuals on the basis of _________________________.– Fragments are generated by restriction

enzymes.– Each individual’s DNA is different enough that

these enzymes will generate different lengths of fragments in two different individuals.

Techniques Used in DNA Fingerprinting

• ________________– A way to separate DNA fragments based on their

length– DNA sample is loaded into a gel matrix and an

electrical current is applied.– _________fragments travel through the gel faster.– Creates a banding pattern of fragments– DNA is negatively charged

• Negative cathode (black)• Positive anode (red)• Run from the smoke into the fire

Gel Electrophoresis

• EtBr is used to bind to the DNA fragments so they fluoresce under UV light

DNA Fingerprinting in a Crime Case

Lane 1: victims DNALane 2: Skin sample from under victims finger nailsLane 3: perp 1 DNA sampleLane 4: perp 2 DNA sampleLane 5: perp 3 DNA sample

What does the blue box represent?What do the black lines represent?Which way did the DNA migrate? Who is the murderer?

1 2 3 4 5

-

+

Genetically modifying organisms

Transform into E. coli Plate on

media with antibiotic

Blue colonies were transformed

++ +Gene coding for antibiotic resistance

Gene coding for Beta galactosidaseCloning vector/ plasmid

Genetic Engineering

Digest plasmid using restriction enzyme and mix with insulin gene

Transform plasmid into a cell

• The bacterial cells now produce a product that can be harvested, purified

This human insulin (recombinant DNA origin) is structurally identical to the insulin produced by the human pancreas. This human insulin is produced by recombinant DNA technology utilizing Saccharomyces cerevisiae (bakers' yeast) as the production organism.

Genetically engineered eukaryotes

• – Same thing as transformation except in eukaryotes

• Genetically engineered plants– Bt-toxin

• Toxin produced by Bacillus thuringiensis• Toxic to insects• Gene incorporated into corn, cotton, and potatoes

– Roundup TM ready crops• Soybeans, corn, cotton• Resist effects of roundup on crops, kills weeds

Probe Technologies

• DNA probes can be used to locate specific nucleotide sequences of DNA or RNA– Single stranded DNA that has a fluorescent dye

FISHprobe technology

• Fluorescence in situ Hybridization– Rapidly identify organisms

• Bypassing the need to grow in a culture• Identify Mycobacterium tuberculosis in sputum

– Using specific probes for TB

Stem Cells

• Stem cells have not completed the determination and differentiation process, so they have the potential to develop into many different cell types.– Stem cells could be used to replace damaged

tissues and organs in humans.– Could be used to cure diseases

• Parkinson’s disease• Diabetes

Embryonic Stem Cells

• Embryonic stem cells come from embryos and have not completed the process of determination.– These have great potential.– Embryos must be destroyed in order to obtain

them and work with them.• Ethical issues

The Culturing of Embryonic Stem Cells

Adult Stem Cells and iPS cells

• Adult stem cells have partially completed the process of determination.– These have less potential; they are already specific

tissue types (blood, bone, etc).– There is no ethical dilemma in obtaining them.

• iPS cells (induced pluripotent stem cells)– Reprogramming mature cells (epithelial or fibroblast)

using transfection– Transfected cells contain plasmid with genes coding

for pluripotency