PCR

DNA Replication and DNA

Polymerase

Polymerase Chain Reaction

Polymerase: DNA polymerase
DNA polymerase duplicates DNA
Before a cell divides, its DNA must be
duplicated
Chain Reaction: The product of a
reaction is used to amplify the same
reaction
Results in rapid increase in the product

DNA polymerase
Duplicates DNA
Necessary for reproduction of
new cells
More than one DNA polymerases
exist in different organisms

Properties of DNA polymearse

Needs a pre-existing DNA to
duplicate
Cannot assemble a new strand
from components
Called template DNA

Can only extend an existing

piece of DNA
5

Called primers

Taq DNA polymerase

Derived from Thermus aquaticus
Heat stable DNA polymerase
Ideal temperature 72C

Thermal Cycling

A PCR machine controls

temperature
Typical PCR go through three
steps
Denaturation
Annealing
Extension

Denaturation
Heating separates the
double stranded DNA
Denaturation

Slow cooling anneals

the two strands
Renaturation

Heat

Cool

Annealing
Two primers are supplied in molar
excess
They bind to the complementary region
As the DNA cools, they wedge between
two template strands
Optimal temperature varies based on
primer length etc.
Typical temperature from 40 to 60 C

Extension

DNA polymerase duplicats DNA

Optimal temperature 72C

PCR Amplification

Exponential Amplification of template DNA

Typical PCR mix

In a thin wall Eppendorf tube assemble
the following
PCR components

Amount

Template DNA (5-200 ng)

1 mM dNTPs (200 uM final)
10 X PCR buffer
25 mM MgCl2 (1.5 mM final)
20 uM forward primer (20 pmoles final)
20 uM reverse primer (20 pmoles final)
5 units/uL Taq DNA polymerase (1.5 units)
Water

variable
10 uL
5 uL
3 uL
1 uL
1 uL
0.3 uL
Variable

Final Volume

50 uL

Polymerase Chain Reaction - PCR

PCR amplifies DNA
Makes lots and lots of copies of a few
copies of DNA
Can copy different lengths of DNA,
doesnt have to copy the whole
length of a DNA molecule
One gene
Several genes
Lots of genes

Artificial process which imitates

natural DNA replication

How PCR Works

Reagents Needed
DNA sample which you want to amplify
DNA polymerase
Taq DNA polymerase Works at high temps
(explained in a minute)
Nucleotides
Called dNTPs
Pair of primers
One primer binds to the 5 end of one of the
DNA strands
The other primer binds to the 3 end of the antiparallel DNA strand
Delineate the region of DNA you want amplified
Water
Buffer

How PCR Works

Protocol
Put all reagents into a PCR tube
Break the DNA ladder down the middle to
create two strands, a 5 to 3 strand and a
3 to 5 strand
Melting or heat denaturation

Bind each primer to its appropriate strand

5 primer to the 5 to 3 strand
3 primer to the 3 to 5 strand
Annealing

Copy each strand

DNA polymerase
Extending

How PCR Works

Temperature Protocol
Initial Melt: 94C for 2 minutes
Melt: 94C for 30 seconds
30-35
Anneal: 55C for 30 seconds
cycles
Extend: 72C for 1 minute
Final Extension: 72C for 6
minutes
Hold: 4C

How PCR Works

Gel Electrophoresis of DNA

Gel electrophoresis detects the
presence of DNA in a sample
Gel electrophoresis detects the number
of nucleotides in a fragment of DNA
e.g., the number of nucleotides in a DNA
region which was amplified by PCR
Is a rough estimate, is not exact, need
more sophisticated sequencing
techniques to get an exact number of
nucleotides
Can be used to tentatively identify a
gene because we know the number of
nucleotides in many genes

How Gel Electrophoresis of DNA

Works
A sample which contains fragments of DNA is forced

by an electrical current through a firm gel which is

really a sieve with small holes of a fixed size
Phosphate group in DNA is negatively charged so it
is moved towards a positive electrode by the current
Longer fragments have more nucleotides
So have a larger molecular weight
So are bigger in size
So arent able to pass through the small holes in the
gel and get hung up at the beginning of the gel
Shorter fragments are able to pass through and
move farther along the gel
Fragments of intermediate length travel to about the
middle of the gel

DNA fragments are then visualized in the gel with a

special dye
The number of nucleotides are then estimated by
comparing it to a known sample of DNA fragments
which is run through the gel at the same time

How Gel Electrophoresis of DNA

Works

Reagents Needed
Sample of DNA fragments
Known sample of DNA fragments
DNA ladder

Gel
Agarose

Dye to visualize the movement of the

sample as it is traveling through the gel
Loading dye Blue juice
So know when to stop so sample doesnt
just run out of the gel

Dye to visualize DNA after it has

traveled to its final spot in the gel
Syber Safe

Buffer

How Gel Electrophoresis of DNA

Works
Equipment Needed
Box to hold the gel
Comb to create small wells in the
agarose gel to put the DNA sample
into at the beginning of the gel
Positive and negative electrodes
to create the electrical current
Power supply
Gel photo imaging system

How Gel Electrophoresis of DNA

Works