Biotechnology learning objectives
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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

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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’


Biotechnology learning objectives

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’


Biotechnology learning objectives

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

-

+

+/-


Central framework of molecular biology

DNA

RNA

Protein

Trait

Central Framework of Molecular Biology


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.


Biotechnology learning objectives

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: AdenineRNA: 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


Biotechnology learning objectives

A gem from 2007


Biotech extended

Biotech Extended…


Elisa antibody structure

Heavy chain

Disulfide bonds

Light chain

ELISA Antibody Structure


Elisa enzyme linked immunosorbant assay

ELISA Enzyme-Linked ImmunosorbantAssay


Elisa kit results

ELISA Kit Results


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


Example pregnancy test

Example: Pregnancy Test


More biotech extended

More Biotech Extended…


Students need to recognize molecular homologies similarities

Students need to recognize molecular homologies/similarities

A great FRQ from 2009


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