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 DNA-
mediated transformation• Be familiar with PCR, RT PCR/Western Blotting,
genomics and ELISA
What Is PCR?
• DNA replication gone crazy in a test tube!
• Makes millions of copies of a target sequence from template DNA
• Uses heat-resistant Taq polymerase from Thermus aquaticus
What Is Needed for PCR?
• Template (the DNA you want to amplify for the study)
• Sequence-specific primers flanking the target sequence:
• Nucleotides (dATP, dCTP, dGTP, dTTP)
• Magnesium ions (enzyme cofactor)
• Buffer, containing salt
• Taq polymerase
3’
5’
5’
3’
3’ 5’
3’5’
Forward primer Reverse primer
Target sequence
How Does PCR Work?
• Heat (94°C) to denature DNA strands
• 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 separate3’
5’
5’
3’
5’
3’
3’
5’
Denaturation of DNA at 94°C
Annealing Primers
• Primers bind to the template sequence• Taq polymerase binds to double-stranded substrate
3’
5’
5’
3’
3’
5’
5’
3’3’ 5’3’5’
Primers anneal at 60°C
Taq Polymerase Extends…
• Taq polymerase extends primer
• DNA is replicated
Extends at 72°C
3’
5’3’ 5’3’5’
3’
5’3’ 5’3’5’
5’
3’
5’
3’
Exact-length Target Product is
Made in the Third Cycle
Which means there’s extra DNA prior to
cycle 3
3’
5’3’5’3’5’
5’
3’
3’
5’5’
3’
5’
3’
5’3’
3’
3’3’
3’5’
5’
5’
5’
Cycle 1
Cycle 2
Cycle 3
Polymerase Chain
Reaction
PCR Results
• The PV92 Alu is dimorphic so there are two possible PCR products: 641 bp and 941 bp
5’5’ 3’3’Alu
Amplified RegionAmplified Region
No insertion: 641 bpNo insertion: 641 bp
300 bp 300 bp Alu Alu insertinsert
641 bp 641 bp
Alu insertion: 941 bp Alu insertion: 941 bp
Actual Alu PCR Results
941 bp941 bp641 bp641 bp
-- +/-+/-++
++ -- +/-+/-
Central Framework of
Molecular Biology
DNA RNA Protein Trait
Protein Size
• Beta Lactamase– Ampicillin resistance
• Green Fluorescent Protein (GFP)
– Aequorea victoria jellyfish gene
• araC regulator protein– Regulates GFP
transcription
Bacterial DNA
Plasmid DNA
Bacterial cell
Genomic DNA
Why Perform Each Transformation
Step?
2. Incubate on iceslows fluid cell
membrane
3. Heat-shockIncreases permeability
of membranes
4. Nutrient broth incubation
Allows beta-lactamase expression
Beta-lactamase(ampicillin resistance)
Cell wall
GFP
There’s an essential component that’s not shown…
There’s lot’s of important stuff here…
Grow? Glow? • 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
+pGLO
Gene Regulation
RNA Polymerase
araC
ara GFP Operon
GFP Gene
araC GFP Gene
araC GFP Gene
Effector (Arabinose)
B A DaraC
B A DaraC
RNA Polymerase
Effector (Arabinose)
araC B A D
ara Operon
Central Dogma of Molecular Biology
DNA
RNA
Protein
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 is composed of nucleotides.
RNA is composed of nucleotides.
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 5’ CCTGACTCCT GTGGAGAAGT CTGCCGTTAC
A ----------------> T DNA
GAG ----------------> GUG RNA
glutamate ----------------> valine protein
Nucleotides:
DNA: Adenine RNA: Adenine Guanine Guanine Cytosine Cytosine
Thymine ----------------> Uracil
Go BACK to figure 17.6 and convince yourself that this works. This will be a big part of the
intro.
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
-CC TNAGG-
Incubate with Bsu36I at 37C
Digestion of beta globin DNA with Bsu36I
Normal beta globin gene(531 base pairs)
CCTGAGG
Sickle beta globin gene(531 base pairs)
CCTGTGG
Incubate with Bsu36I
(331 base pairs) (200 base pairs) (531 base pairs)
Incubate with Bsu36I
+
AAuncut
DNAladder
1000 bp
500 bp
AAcut
ASuncut
AScut
SScut
SSuncut
Analysis of globin DNA by Gel Electrophoresis, following Bsu36I digestion
AA: homozygous for normal gene
AS: heterozygous (trait)
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
A gem from 2007
Biotech Extended…
ELISA Antibody Structure
Light chain
Heavy chain
Disulfide bonds
ELISA Enzyme-Linked
ImmunosorbantAssay
ELISA Kit Results
Real-world Applications of
Antibodies
Uses– Disease diagnosis– Basic Research – Immunotherapy
Applications– Dipstick tests/ELISA
– Immunostaining– Western blotting
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
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