Lab 9. Human Mitochondrial Analysis using
PCR and Electrophoresis
Major Goals of this Experiment
•
Isolate mitochondrial DNA (mtDNA) from cheek cells
and amplify two separate regions of the mtDNA using the
polymerase chain reaction (PCR).
•
Following PCR, use electrophoresis to separate and
determine the size of the PCR amplified mtDNA
fragments
•
Compare DNA polymorphisms between individuals in the
class
•
Understand the structure and function of mitochondria
Overview of the Procedure (page 6)
Day 1 Activities (Monday 11/30/09)
• Module I: Isolation of mtDNA from human hair follicles or cheek cells: Pages 7 - 8
• Module II: Amplification of two regions of mtDNA by PCR: Pages 9 - 10
Day 2 Activities (Tuesday 12/01/09)
• Module III: Separation of PCR amplified mtDNA regions by Electrophoresis:
pages 11 - 14
Day 3 Activities (Friday 12/04/09)
• Module IV: Stain Gels and Visualization of the PCR amplified mtDNA regions:
Page 15
• Size Determination of the of the PCR amplified mtDNA regions—done at home:
Page 16
Genetic Map of mtDNA
mt DNA—a circular chromosome!
•
16,569 base pairs
•
37 genes that encode for
0 / 16569 bp
 ETC complex proteins
12360 bp
 Mitochondrial ribosomal RNA
 ATP Synthase
PCR
Products
11688 bp
• The D-loop
 Varies greatly between individuals
9199 bp
8278 bp
 Can be sequenced to demonstrate
variations.
 Cannot be used to conclusively link
suspects to crime scenes
 Used to include or exclude suspects
for further scrutiny.
Fig. 3 (page 2). Genetic Map of mtDNA
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
PCR
Polymerase
Chain Reaction
(page 5)
Three cycles of the
polymerase chain
reaction
Gel Electrophoresis
1. A method of separating mixtures of large molecules (e.g.
proteins and DNA & RNA fragments) 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
The 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