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Biotechnology Learning Objectives
• Be able to describe the components of DNA
electrophoresis, and recognize patterns in a gel
• Be able to describe the form and function of restriction
enzymes (restriction endonucleases)
• Be able to describe the process of DNA-mediated
transformation of bacterial cells
• Discuss the molecular basis for the results of a DNAmediated transformation
• Be familiar with PCR, RT PCR/Western Blotting,
genomics and ELISA
• DNA replication gone crazy
in a test tube!
What Is PCR?
• Makes millions of copies of
a target sequence from
template DNA
• Uses heat-resistant Taq
polymerase from Thermus
aquaticus
• Template (the DNA you want to amplify for the study)
• Sequence-specific primers flanking the target
sequence:
5’
3’
What Is Needed
for PCR?
3’
Forward primer
5’
5’
3’
Reverse primer
5’
3’
Target sequence
• Nucleotides (dATP, dCTP, dGTP, dTTP)
• Magnesium ions (enzyme cofactor)
• Buffer, containing salt
• Taq polymerase
• Heat (94°C) to denature DNA
strands
How Does PCR
Work?
• Cool (60°C) to anneal primers to
template
• Warm (72°C) to activate Taq
polymerase, which extends primers
and replicates DNA
• Repeat multiple cycles
Denaturing
Template DNA
Heat causes DNA strands to separate
5’
3’
3’
5’
Denaturation of DNA at 94°C
5’
3’
3’
5’
• Primers bind to the template sequence
• Taq polymerase binds to double-stranded substrate
3’
5’ Annealing
3’
5’
Primers
Primers anneal at 60°C
5’
3’
5’
3’
3’
5’
3’
5’
• Taq polymerase extends primer
• DNA is replicated
Taq Polymerase
Extends…
3’
5’
5’
3’
3’
5’
3’
5’
Extends at 72°C
5’
3’
3’
5’
3’
3’
5’
5’
Cycle 1
Exact-length
Target Product is
Made in the
Third Cycle
Which means
there’s extra
DNA prior to
cycle 3
5’
5’
3’
3’
5’
3’
3’
5’
Cycle 2
5’
3’
3’
5’
3’
5’
3’
5’
Cycle 3
3’
5’
3’
5’
5’
3’
5’
3’
Polymerase
Chain
Reaction
No insertion: 641 bp
• The PV92 Alu is
dimorphic so
there are two
possible PCR
PCR Results
products:
641 bp
and 941 bp
5’
Alu insertion: 941 bp
300 bp Alu insert
641 bp
Alu
Amplified Region
3’
Actual Alu PCR
Results
+
941 bp
641 bp
+
- +/-
-
+/-
Central
Framework of
DNA
Molecular RNA
Biology
Protein
Trait
• Beta Lactamase
– Ampicillin resistance
• Green Fluorescent
Protein (GFP)
– Aequorea victoria
Protein
jellyfishSize
gene
• araC regulator
protein
– Regulates GFP
transcription
Bacterial cell
Bacterial DNA
Plasmid DNA
Genomic DNA
There’s lot’s of important stuff here…
Why Perform Each
Transformation
Step?
Cell wall
GFP
2. Incubate on ice
slows fluid cell
membrane
3. Heat-shock
Increases permeability
of membranes
4. Nutrient broth
incubation
Allows beta-lactamase
expression
Beta-lactamase
(ampicillin
resistance)
There’s an essential component that’s not shown…
Grow?
Glow?
-pGLO
•
These are important Questions…
•
What’s the role of the LB (-pGLO) plate?
•
On which plates will colonies grow?
– It depends on what you plated
– What traits will be evident?
– Why?
•
Which colonies will glow?
– Why?
-pGLO
+pGLO
+pGLO
ara GFP Operon
ara Operon
ara
C
B
A D
araC
Effector (Arabinose)
Effector (Arabinose)
Gene Regulation
araC
B A D
araC
RNA Polymerase
araC
B A D
GFP Gene
GFP Gene
RNA Polymerase
araC
GFP Gene
Central Dogma of Molecular Biology
RNA is synthesized using
DNA as the template in a
process called
Transcription.
Protein is synthesized from
the information in RNA in a
process called Translation.
DNA
DNA is composed
of nucleotides.
RNA
RNA is composed
of nucleotides.
Protein
Protein is composed
of amino acids.
SS Sickle cell disease is caused by a single base mutation in the
gene that codes for the beta globin subunit of hemoglobin.
Normal beta globin gene 5’ CCTGACTCCT
GAGGAGAAGT CTGCCGTTAC
Sickle beta globin gene
GTGGAGAAGT CTGCCGTTAC
5’ CCTGACTCCT
A
---------------->
T
DNA
GAG
---------------->
GUG
RNA
glutamate ---------------->
valine
Nucleotides:
DNA: Adenine
Guanine
Cytosine
Thymine
RNA: Adenine
Guanine
Cytosine
----------------> Uracil
Go BACK to figure 17.6 and convince yourself
that this works. This will be a big part of the
intro.
protein
DNA can be analyzed by digestion with restriction
enzymes
Restriction enzymes are proteins that cut DNA at specific nucleotide sequences.
The restriction enzyme Bsu36I cuts DNA with the sequence CC^TNAGG.
CCTNAGG
Incubate with Bsu36I at 37C
-CC
TNAGG-
Digestion of beta globin DNA with Bsu36I
Normal beta globin gene
(531 base pairs)
CCTGAGG
Incubate
with Bsu36I
Sickle beta globin gene
(531 base pairs)
CCTGTGG
Incubate
with Bsu36I
+
(331 base pairs)
(200 base pairs)
(531 base pairs)
Analysis of globin DNA
by Gel Electrophoresis, following Bsu36I digestion
_
DNA
ladder
AA
uncut
AA
cut
AS
uncut
AS
cut
SS
uncut
SS
cut
1000 bp
AA: homozygous for normal gene
AS: heterozygous (trait)
500 bp
SS: homozygous for sickle gene
RFLP!
+
Types of Questions, students are
likely to encounter (MCQ)
• Reading a gel/Recognizing patterns
• Applying concept of RFLP to genetic
disorder, paternity cases
• Linking experimentally derived genetic
information to a cladogram
• Describing process of transformation and
describing its utility
Biotech Extended…
ELISA
Antibody
Structure
Heavy
chain
Disulfide
bonds
Light
chain
ELISA EnzymeLinked
Immunosorbant
Assay
ELISA Kit
Results
Uses
– Disease diagnosis
– Basic Research
– Immunotherapy
Applications
– Dipstick tests/ELISA
– Immunostaining
– Western blotting
Real-world
Applications of
Antibodies
Bio-Rad’s HIV-2
ELISA Kit
Bio-Rad’s HIV
Western Blot Kit
Example:
Pregnancy Test
More Biotech Extended…
Students need to recognize
molecular homologies/similarities
A great FRQ from 2009