Lab 8. RT-PCR
A Model for the Molecular Biology of HIV Replication
Major Goals of this Experiment
• To gain an understanding and hands-on experience of the
principles and practice of each of the following:
• Reverse Transcriptase-based Polymerase Chain Reaction
(RT-PCR) and to relate these reactions to HIV replication
• Use of Electrophoresis to separate and determine the size
of the RT-PCR amplified DNA fragment.
• Learn the Molecular biology of HIV life cycle
RT-PCR Lab: Overview of the Procedure
 Day 1 Activities:
o Module I: Production of cDNA by RT-PCR Reaction
o Module II: Pour Gel and Preparation of Gel for Electrophoresis (Steps
1-15)—store gels under buffer in the refrigerator until day 2.
 Day 2 Activities:
o Complete Module II: Load Gels with DNA samples produced by RTPCR and carryout Electrophoresis to separate the DNA fragments.
o Time permitting: Stain gel and visualization the DNA in gel.
 Day 3 Activities:
o Stain gel and visualization the DNA in gel (if not done day 2)
o Module III: Size Determination of the RT-PCR fragments
Importance of Viruses
1. Cause many diseases
•
Viruses easily controlled with a vaccine
 Mumps, Measles, Smallpox, Polio
•
Viruses difficult to control with a vaccine (retroviruses)
 Retroviruses (RNA  DNA)
 Common cold, Influenza (Flu), HIV
2. Used as vectors in biotechnology
•
Used to insert therapeutic genes into a host cell
chromosome
Comparing the size of a virus, a bacterium, and a eukaryotic cell
Viral Structure
1. Nucleic Acid + Protein Coat (Capsid)
•
Some viruses with Membrane (envelope) surrounding capsid

•
Envelope derived from plasma membrane of host cell
No organelles
•
Obligate intracellular parasite
•
Lacks metabolic enzymes, ribosomes, mitochondria
•
Alone, can only infect host cell
2. Nucleic Acid: DNA or RNA
•
Single or Double Stranded
•
4 genes to a few hundred
Viral structure
Classes of Animal
Viruses, Grouped
by Type of
Nucleic Acid
HIV infection
HIV Life Cycle
HIV is a retrovirus:
ssRNA  dsDNA
Function of Reverse
Transcriptase?
Proofreading?
PCR—Polymerase Chain Reaction
1. Quick, easy, automated method to make copies
of a specific segment of DNA
2. What’s needed….
•
DNA primers that “bracket” the desired sequence to
be cloned
•
Heat-resistant DNA polymerase
•
DNA nucleotides
The polymerase
chain reaction (PCR)
Gel Electrophoresis
1. A method of separating mixtures of large molecules
(such as DNA fragments or proteins) on the basis of
molecular size and charge.
2. How it’s done
•
An electric current is passed through a gel containing the
mixture
•
The each molecule travels through the gel is inversely related
to its size and electrical charge:
Rate a 1 / size & charge
•
Agarose and polyacrylamide gels are the media commonly
used for electrophoresis of proteins and nucleic acids.
Gel electrophoresis of macromolecules
Process of DNA
electrophoresis
Step 1
Prepare a tray to hold
the gel
Step 2: Pouring the Gel
A "gel comb" is used to
create “wells” (holes in
the gel to hold the
mixture of DNA
fragments.
Step 2: Pouring the Gel
a.
The gel comb is placed in the
tray.
b.
Agarose powder is mixed with
a buffer solution, The solution
is heated until the agarose is
dissolved—like making Jello
c.
The hot agarose solution is
poured into the tray and
allowed to cool.
d.
After the gel is cooled and
solidified, the comb is removed
and the gel tray is placed in an
electrophoresis chamber.
Step 3: Loading the Gel
a.
Fill electrophoresis chamber
with buffer, covering the gel to
allow electrical current from
poles at either end of the gel to
flow through the gel.
b. DNA samples are mixed with
a "loading dye".
c.
The loading dye

allows you to see the DNA as
you load it and contains
glycerol to make the DNA
sample dense so that it will
sink to the bottom of the well.
Step 4: Running the Gel
a.
A safety cover is placed over the
gel (to keep you from frying
yourself) and electrodes are
attached to a power supply. High
voltage is applied.
b.
DNA fragments migrate through
the gel at various rates,
depending on their size and
c.
When the loading dye reaches
the end of the gel, the current is
turned off, the gel removed from
the try and then developed to see
the DNA fragments
Step 5: Visualization of the DNA Fragments
Stain gel with dye that binds to DNA
a.
Methylene blue (safe)
• Time consuming
• Poor resolution
• View with naked eye
b.
Ethidium Bromide (carcinogen)
• Fast + high resolution
• View under uv-light
• Take Polaroid picture