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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)

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biotechnology learning objectives
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
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

3’

5’

5’

3’

Forward primer

Reverse primer

3’

5’

5’

3’

Target sequence

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
how does pcr work
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
Denaturing Template DNA

Heat causes DNA strands to separate

5’

3’

5’

3’

Denaturation of DNA at 94°C

3’

5’

3’

5’

annealing primers

5’

3’

5’

3’

5’

3’

3’

5’

Annealing Primers

• Primers bind to the template sequence

• Taq polymerase binds to double-stranded substrate

3’

5’

3’

5’

Primers anneal at 60°C

taq polymerase extends
Taq Polymerase Extends…

• Taq polymerase extends primer

• DNA is replicated

3’

5’

5’

3’

5’

3’

5’

3’

Extends at 72°C

5’

3’

5’

3’

5’

3’

3’

5’

slide10
Exact-length Target Product is Made in the Third Cycle Which means there’s extra DNA prior to cycle 3

Cycle 1

5’

3’

5’

3’

5’

3’

3’

5’

Cycle 2

3’

5’

5’

3’

3’

5’

3’

5’

Cycle 3

3’

5’

3’

5’

5’

3’

5’

3’

slide11

Polymerase

Chain

Reaction

pcr results
PCR Results

• The PV92 Alu is dimorphic so there are two possible PCR products: 641 bp and 941 bp

No insertion: 641 bp

Alu insertion: 941 bp

300 bp Alu insert

641 bp

3’

5’

Alu

Amplified Region

actual alu pcr results
Actual Alu PCR Results

-

+/-

+

941 bp

641 bp

-

+

+/-

protein size
Protein Size
  • Beta Lactamase
    • Ampicillin resistance
  • Green Fluorescent Protein (GFP)
    • Aequorea victoria jellyfish gene
  • araC regulator protein
    • Regulates GFP transcription
bacterial dna
Bacterial DNA

Bacterial cell

Plasmid DNA

Genomic DNA

why perform each transformation step
Why Perform Each Transformation Step?

There’s lot’s of important stuff here…

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

LB/Amp

LB/Amp

LB/Amp/Ara

LB

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

ara GFP Operon

ara Operon

araC

GFP Gene

araC

B

A

D

Effector(Arabinose)

Effector (Arabinose)

araC

B

A

D

araC

GFP Gene

RNA Polymerase

RNA Polymerase

B

A

D

araC

araC

GFP Gene

Gene Regulation
central dogma of molecular biology

DNA

RNA

Protein

Central Dogma of Molecular Biology

DNA is composed of nucleotides.

RNA is synthesized using DNA as the template in a process called Transcription.

RNA is composed of nucleotides.

Protein is synthesized from the information in RNA in a process called Translation.

Protein is composed of amino acids.

slide22
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

CCTNAGG

-CC

TNAGG-

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.

Incubate with Bsu36I at 37C

digestion of beta globin dna with bsu36i

CCTGTGG

CCTGAGG

Incubate with Bsu36I

Incubate with Bsu36I

Digestion of beta globin DNA with Bsu36I

Normal beta globin gene

(531 base pairs)

Sickle beta globin gene

(531 base pairs)

+

(531 base pairs)

(331 base pairs)

(200 base pairs)

analysis of globin dna by gel electrophoresis following bsu36i digestion
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)

SS: homozygous for sickle gene

500 bp

RFLP!

+

types of questions students are likely to encounter mcq
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
elisa antibody structure

Heavy chain

Disulfide bonds

Light chain

ELISA Antibody Structure
real world applications of antibodies

Applications

    • – Dipstick tests/ELISA
    • – Immunostaining
    • – Western blotting
Real-world Applications of Antibodies

Uses

– Disease diagnosis

– Basic Research

– Immunotherapy

Bio-Rad’s HIV-2

ELISA Kit

Bio-Rad’s HIV

Western Blot Kit

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