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The polymerase chain

reaction (PCR)
Experiment Goals
Understand how PCR technique works
Perform PCR experiment
Analyze PCR products
What is PCR?
Definition
The polymerase chain reaction (PCR) is a
technique to amplify a piece of DNA very
rapidly outside a living cell
Development of PCR
1971: Khorana described basic principle
of DNA replication using DNA primers
1983: Dr. Karry Mullis developed PCR
technique, for which he received the
Nobel Prize in Chemistry in 1993.
PCR Applications
PCR is now a common and often indispensable
technique used in medical and biological research labs
for a variety of applications.

Structural analysis Mapping


DNA typing Site-directed mutagenesis
Disease detection Sequencing
Cloning Forensic medicine
Mutation analysis Scientific research
Detection of gene Pre-natal diagnosis
expression
How does PCR work?
DNA is denatured (H-bonds are broken
between strands of DNA with heat), 94oC
Primers attach to complementary sequences
of single stranded DNA, 55-60oC
DNA polymerase attaches to primer with
ssDNA and extends DNA fragment, 72oC
Thus, making double stranded DNA
This is done by changing the temperature
PCR Reaction Components

1) Target DNA - contains the sequence to be amplified.

2) Pair of Primers - oligonucleotides that define the sequence


to be amplified.
3) dNTPs - deoxynucleotidetriphosphates: DNA building blocks.

4) Thermostable DNA Polymerase - enzyme that


catalyzes the reaction
5) Mg++ ions - cofactor of the enzyme

6) Buffer solution maintains pH and ionic strength of


the reaction solution suitable for the activity of the
enzyme
1) Target DNA
Target of DNA can be
a single gene
part of a gene
or a non-coding sequence
DNA Quality
DNA should be intact and free of
contaminants that inhibit amplification.
Contaminants can be purified from the
original DNA source.
Heme from blood, and melanin from hair
Contaminants can be introduced during the
purification process.
Phenol, ethanol, sodium dodecyl sulfate (SDS)
and other detergents, and salts.
DNA quantity
More template is not necessarily better.
Too much template can cause nonspecific
amplification.
Too little template will result in little or no
PCR product.
How Big A Target is?
Amplification products are typically in the
size range 100-1500 bp.
Longer targets are amplifiable >25 kb.
Requires modified reaction buffer,
cocktails of polymerases, and longer
extension times.
2) Pair of Primers
Primers define the DNA sequence to be
amplifiedthey give the PCR specificity.
Primers bind (anneal) to the DNA template and
act as starting points for the DNA polymerase,
DNA polymerases cannot initiate DNA synthesis without
a primer.
The distance between the two primers
determines the length of the newly synthesized
DNA molecules.
3) dNTPs
(deoxynucleotidetriphosphates)
The building blocks for the newly
synthesized DNA strands.
dATP, dGTP, dCTP or dTTP
4) Thermostable DNA Polymerase
DNA Polymerase is the enzyme responsible for
copying the sequence starting at the primer from
the single DNA strand
Commonly use Taq, an enzyme from the
hyperthermophilic organisms Thermus aquaticus,
isolated first at a thermal spring
This enzyme is heat-tolerant
it is thermally tolerant (survives the melting T of DNA
denaturation)
which also means the process is more specific, higher
temps result in less mismatch more specific
replication
Running PCR
The PCR is commonly carried out in a reaction volume
of 15-100 l in small reaction tubes (0.2-0.5 ml
volumes) in a thermal cycler.
The thermal cycler allows heating and cooling of the
reaction tubes to control the temperature required at
each reaction step.
Thin-walled reaction tubes permit favorable thermal
conductivity to allow for rapid thermal equilibration.
Most thermal cyclers have heated lids to prevent
condensation at the top of the reaction tube.
Older thermocyclers lacking a heated lid require a
layer of oil on top of the reaction mixture or a ball of
wax inside the tube.
Initialization step
Prior to the first cycle, there is an
initialization step
the PCR reaction is often heated to a
temperature of 94-96C, and this
temperature is then held for 1-9 minutes
This first hold is employed to ensure that
most of the DNA template and primers are
denatured,
Also, some PCR polymerases require this
step for activation
PCR Reaction Cycles
One PCR cycle consists of a DNA
denaturation step, a primer annealing step and
a primer extension step.
DNA Denaturation: Expose the DNA template to
high temperatures to separate the two DNA
strands and allow access by DNA polymerase and
PCR primers.
Primer Annealing: Lower the temperature to
allow primers to anneal to their complementary
sequence.
Primer Extension: Adjust the temperature for
optimal thermostable DNA polymerase activity to
extend primers.
PCR
PCR: First 4 Cycles
PCR: Completed Amplification Cycle
PCR: Completed Amplification Cycle

Each cycle: 1 copy of DNA template will


give 2 copies from double-stranded DNA
templates.
n cycles will give 2n copies
Assuming a cycle lasts 6 min:
1 double-stranded DNA molecule
35 cycles
34x109 copies in 3.5 hrs!
There are also 60 other DNA copies
DNA copies vs Cycle number

2500000

2000000

DNA1500000
copies

1000000

500000

0
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

Cycle number
Analyze PCR products
Check a sample by gel electrophoresis.
Is the product the size that you
expected?
Is there more than one band?
Is any band the correct size?
May need to optimize the reaction
conditions.
PCR Modifications
Nested PCR
increases the specificity of DNA amplification, by reducing
background due to non-specific amplification of DNA. Two sets
of primers are being used in two successive PCRs.
Multiplex PCR
The use of multiple, unique primer sets within a single PCR
mixture to produce amplicons of varying sizes specific to
different DNA sequences.
Reverse-transcriptase PCR
(Reverse Transcription PCR) is a method used to amplify,
isolate or identify a known sequence from a cellular or tissue
RNA. The PCR is preceded by a reaction using reverse
transcriptase to convert RNA to cDNA.
Polymerase Chain Reaction
Controls for PCR
Blank reaction (Negative control reaction)
Controls for contamination
Contains all reagents except DNA template

Positive control reaction


Controls for sensitivity
Contains all reagents and a known target-containing
DNA template
Contamination of PCR
Reactions
Most common cause is carelessness and bad
technique.
Separate pre- and post-PCR facilities.
Dedicated pipettes and reagents.
Change gloves.
Aerosol barrier pipette tips.
10% bleach, UV light
Procedure
1- Prepare master Mix
2- Program the thermocycler
3- Run the samples on thermocycler
4- Analysis of PCR products
Target DNA
Amplification of part of the Human
growth hormone gene
Specific primers used
Forward primer: 5-
TCCCTTCCCAACCATTCCCTTA-3
Reverse primer: 5-
CCACTCACGGATTTCTGTTGTGTTTC-
3
1- Master Mix
PCR reaction mixture
Reagent Volume (l) Final concentration
PCR buffer (X10) 2.0 10 mM
MgCl2 (25 mM) 1.6 2.0 mM
dNTPs (100mM) 0.1 0.1 mM
Primer 1 (F) 0.2 1.0 M
Primer 2 (R) 0.2 1.0 M
Taq DNA
0.25 2.0 U
polymerase
DNA template 2.0 100 ng
Water 13.7
2- Program the Thermocycler
The Thermocycler Profile is:
Step 1: Denaturation for 3 min. at 95oC
Step 2: 35 cycles
Melting for 60 sec. at 95oC
Annealing for 60 sec. at 57oC
Extension for 90 sec. at 72oC
Step 3: Final elongation for 10 min. at
72oC
4- Analysis of PCR products

Analyse products on 2%
-ve +ve Sample
agarose gel containing
ethidium bromide
Visualize the PCR product
on UV transilluminator