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