Date post: | 01-Jan-2016 |
Category: |
Documents |
Upload: | cora-banks |
View: | 221 times |
Download: | 3 times |
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