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Laboratory work Nr.2
PCR
Polymerase Chain Reaction
Polymerase chain
reaction (PCR)
 PCR discovery by Kary Mullis in 1983 allows
the scientists to mimic the cell’s own natural DNA
replication process in a test tube.
 For PCR discovery Mullis received the Nobel prize in
Chemistry in 1993.
 PCR allows the amplification of specific DNA sequences in a
large quantities from incredibly small amounts of material.
 This method is so original and significant, that virtually divides
biology into the two epochs of before PCR and after PCR.
 PCR become a central laboratory method in molecular
biology.
DNA replication in vivo
The majors steps in DNA replication
process in cells :
1. Helicase unwinds the DNA helix
2. RNA primase synthesize
complementary RNA primers
3. DNA polymerase attach to the primer
ends
4. Synthesis of complimentary DNA
fragment.
DNA replication in vitro
1. dsDNA denaturation at 96⁰C
2. Synthetic DNA primers bind to
DNA strand
3. DNA polymerization reaction
DNA
pol
DNA
pol
DNA polymerase is a protein.
DNA
pol
Proteins tend to denature (unfold and
lose activity) at high temperatures.
Therefore, in PCR we use thermostable
DNA polymerases.
Thermostable DNA polymerases
• Isolated from the hot spring organisms.
• Taq DNA polymerase was originally isolated from thermophilic bacterium
Thermus aquaticus that lives in hot springs were temperatures exceed
+70⁰C (found in Yellowstone national park). Halflife of Taq DNA polymerase
at +95⁰C is 1.6 hours.
• Pfu DNA polymerase was isolated from hiperthermophilic arhaebacterium
Pyrococcus furiosus (Volcanic island in Italy) that lives at +70- +103⁰C
temperatures.
• Taq polymerase is faster and cheaper than Pfu polymerase.
• Pfu polymerase have the lowest error rate.
In vivo vs In vitro DNA
replication
The majors steps in DNA replication
process in vivo
The majors steps in DNA replication
process in vitro
Helicase unwinds the DNA helix
DNA unwinding occurs at high
temperature
RNA primase synthesize
complementary RNA primers
Synthetic DNA primers
DNA polymerase attach to the primer DNA polymerase attach to the primer
ends
ends
Synthesis of complimentary DNA
Synthesis of complimentary DNA
fragment.
fragment.
PCR
DNA in the PCR reaction
would grow exponentially.
25 to 35 cycles is the standard
for a PCR reaction.
This results in from approximately 34
million to 34 billion copies of desired
sequence, respectively.
PCR primers
• Synthetic single strand DNA fragments (16-25nt) that are
complimentary to template DNA.
• Each primer has melting temperature (Tm). Tm relies on length and
composition of the primers.
• Praimers annealing temperature should be 5⁰C below the lowest
primer Tm.
• Tm of the primers could be calculated by using formula
• Tm = 2 X (A+T) + 4 X (G+C)
PCR applications
PCR is widely used in medical and biological research
for a variety of applications
• DNA amplification for DNA cloning and sequencing
• For scientific and analytical objectives – genome analysis,
artificial DNA construct analysis
• Clinical diagnostics – detection of patogen presence in sample
• Forensic medicine – to reveal the person’s identity, paternity
tests
• Food product analysis (example, if the meat product contains
horse or cat meat)
PCR laboratory work
Target substrates
Diabetes caused by mutations in the HNF1A (encoding hepatocyte nuclear
factor-1 alpha) and GCK4 (encoding glucokinase 4) genes is one of the
most common types of maturity onset diabetes of the young (MODY).
HNF1α is a transcription factor that is important for the normal development
of beta cells. Mutations in the HNF1A gene cause diabetes by lowering the
amount of insulin that is produced by the pancreas. Mutations in HNF1A
accounts for 70% of MODY cases.
GCK4 is a gene that codes for an enzyme, known as glucokinase, which
helps the body produce insulin in response to increases in blood sugar.
Mutations in one of the two copies of the GCK4 gene result in blood sugars
that are mildly elevated above normal levels. This is one of the most
common forms of MODY, it is estimated to affect about 1 in every 1000
people.
PCR procedure
1. Setting up the reaction mix
PCR 1 tube
GCK4 gene
fragment
amplification
PCR 2 tube
HNF1A gene
fragment
amplification
PCR 3 tube
Negative controlno template
control (NTC)
Polymerase chain reaction
Procedure
1. Set up the reaction mixture:
Buffer
Water
Primer Forward
Mg2+ ions
5’- ATTGCCA-3’
ATTGCCA
Primer Reverse
dNTP mix
5’- TATCCGA-3’
Template DNA
dATP+ dCTP + dGTP
+ dTTP
DNA polymerase
Setting up the reaction mix
1. Place 0.2ml PCR tubes on ice.
2. Set up 25 µl PCR reaction (keep all your reagents on ice).
Reagents
PCR 1
volume, µl
PCR 2
volume, µl
PCR 3, NTC
volume, µl
1x
2.5mM
200 µM
Sterile dH2O
10x Hot Fire DNA buffer
2.5
2.5
17.4
2.5
MgCl2 (25mM)
dNTP mix (10mM)
2.5
0.5
2.5
0.5
2.5
0.5
Primer Fw (100 pmol/µl)
0.8
0.8
0.8
Primer Rev (100 pmol/µl)
0.8
0.8
0.8
Template DNA
Hot
FirePol
DNA
Polymerase (5U/µl)
Total volume of reaction
Final
concentration
-
50 ng
2.5U
0.5
0.5
0.5
25 µl
25 µl
25 µl
•In PCR1 tube the primers 90 and 91 should be used for amplification of the GCK4 gene fragment.
•In PCR2 tube the primers S19L Fw and S19L Rev should be used for amplification of the HNF1A gene fragment.
•In PCR3 tube you can choose one primer set or another.
PCR reaction
3. Set up the PCR program
Initial Denaturation for 10 minutes at 95°C:
In this initiation step the hydrogen bonds are broken between the
nucleotide base pairs and DNA strands separate from each other.
Denature 30 seconds at 95°C: Continued denaturation of DNA
double helix.
PCR reaction
3. Set up the PCR program
Anneal primers for 30 seconds at 60°C:
The forward and reverse primers anneal to each of the single stranded
DNA template strands. The DNA polymerase bind to the primer
DNA sequence.
Extend DNA for 30 seconds at 72°C: The Taq polymerase has an optimal
temperature around 70-75°C so this step enables the DNA polymerase to
synthesize and elongate the new target DNA strand accurately and rapidly.
Repeat steps 2-4 35 times.
PCR reaction
3. Set up the PCR program
Final Extension for 7 minutes at 72°C: A final extension to fillin any protruding ends of the newly synthesized strands.
4. Place the PCR tubes into PCR thermocycler and run
the program.
Validating the PCR reaction
Once your PCR reaction has run (2h), you will determine success or failure.
You will take some of the final PCR reaction and run it out on an agarose
gel with an appropriate molecular weight marker to make sure
 that the reaction was successful and
 the amplified product is the expected size relative to the maker.
5. Add 5 µl of 6x Loading dye into each PCR reaction tube and vortex.
Carefully load your samples into the wells of the 1% agarose gel
and run the gel.
PCR result analysis by agarose gel
electrophoresis
4) PCR reaction analysis in agarose gel
100bp marker
Electrophoresis uses an electrical field to move the negatively
charged DNA toward a positive electrode through an agarose gel matrix.
The gel matrix allows shorter DNA fragments to migrate more quickly than larger ones.
1.
2.
3.
4.
5. -cont.
Thus, you can
accurately
determine the length
of a DNA segment
by running it on an
agarose gel
alongside a DNA
ladder (a collection
of DNA fragments of
known lengths).
Genomic DNA concentrations
Genomic DNA concentration and purity
 DNA yield and purity could be estimated by measurement of
absorbance.
 DNA concentration is estimated by measuring the absorbance at
260nm (A260), adjusting the A260 measurement for turbidity (A320
measurement), multiplying by the dilution factor, and using the
relationship that
A260 of 1.0= 50 µg/ml pure double stranded DNA.
 DNA purity calculate as the ratio of the absorbance at 260nm
divided by the readings at 280nm.
Good quality DNA will have an A260/A280 ratio 1.7-2.0.
A reading below 1.7 does not render that DNA unsuitable for any application,
but lower ratios indicate more contaminants are present.
Your genomic DNA extraction
results
Genomic DNA concentrations
Tube
number
DNA conc, ng/µl
DNA purity, A260/A280
1.
58
1.78
2.
79
1.4
3.
22
1.96
4.
379
1.68
5.
25
1.33
6.
77
1.77
7.
112
1.7
8.
12
1.85
9.
89
1.84
10.
63
1.86
31
1.5
11.
12.
The ideal purity for genomic DNA is in the range 1.80 – 1.89.