Amplification of DNA by
Polymerase Chain Reaction
(PCR)
Prepared by:
kholoud Al- Homoudi
Molecular Biology Unit
Central Lab
King Saud University
1
The Polymerase Chain Reaction (PCR) and Its Applications
I.
II.
III.
Definition of PCR
Requirements for PCR
PCR Process
A. Denaturing Stage
B. Annealing Stage
C. Extending Stage
D. Replication
The Polymerase Chain Reaction (PCR) is an in vitro method to amplify a specific region of
DNA. PCR is extremely sensitive, with the capability of amplifying minuscule quantities of
DNA.
REQUIREMENTS
1. DNA sample
· very small amounts (ng or sometimes less) if DNA is in good shape
· may be able to use DNA from only one cell
2. Two primers
· flank region you are interested in
· you must know the sequence of the flanking regions so you can order appropriate
primers
2. Heat stable polymerase.
3. Four d NTPs.
4. Thermocycler (standard, but optional)
Changes temperature very rapidly for each cycle. (denature, anneal, extend).
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Amplification of DNA by polymerase chain reaction (PCR).
Principle:
The PCR is an in vitro enzymatic synthesis and amplification of specific DNA sequences.
PCR mimics the in vivo process of DNA replication. It begins with one molecule of the
double-stranded DNA, using specific primers that flank both sides of the DNA to be
amplified. In order for the primer to work on the DNA template and amplify it, it needs a
set of certain buffer, temperature degrees, and building blocks for the new synthesized
fragments (dNTPs = deoxy nucleotide tri-phosphate), as well as the very important enzyme
that will carry out the amplification, or the polymerisation of the DNA template. The
number of copies of the DNA fragment rises exponentially, since every newly synthesized
DNA-sequence is also a matrix for the next copy. For PCR, the following is needed:
(i) A thermostable DNA-polymerase: The most used polymerase is the Taqpolymearase. It is isolated from the bacteria Thermophilus aquaticus which
occurs in hot springs. This enzyme is used for the PCR reaction, where the
DNA is being denatured at a temperature of 95 °C, whereas the DNA
polymerase remains stable.
(ii) The template-DNA: The template-DNA can consist of very different DNA’s. For
example: genomic DNA libraries and cDNA libraries. In this study genomic DNA
extracted from Saudi Individuals was used (or genomic DNA).
(iii) Oligonucleotides (primers): Primers are short, single-stranded sequences of mostly 2025 nucleotides length, which link with another single-stranded matrix by complimentary
base pairing. They serve as the starting point for the synthesis of the complimentary strand
of the DNA-polymerase.
(iv) Nucleotides (dNTPs): dNTPs are the units used for
the synthesis of the new DNA-
strand. With the help of the DNA-polymerase they are attached to the end of the primer
complimentary to the matrix, thus creating the DNA-daughter strand.
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In Principle, the PCR-reaction is subdivided into three steps:
1) Denaturation: During denaturation, the template DNA is separated (denatured) into its two
separate strands by heating at the temperature about 95º C.
2) Annealing: This involves the annealing of the primer to the denatured.
3) Extension: The third step, the synthesizing, takes place at a temperature of around 72º C.
This corresponds to the optimal temperature for the Taq- polymerase to work. The
polymerase prolongs the paired short oligonucleotides (primers) according to the DNAmatrix, until the double-stranded DNA-molecule is complete again. The three steps are
repeated in cycles, usually 30-35 times as an exponential increase occurs, following the 30-35
cyc les that, there is enough DNA to be detected and ana lyzed.
DNA. The temperature is lowered to a degree specific for the primer, which generally lies
between 40ºC and 68ºC. This guarantees that the primer takes its place at the specific DNArecognition-sequence (according to their complementary bases) of the single-stranded
DNA- template-sequence.
Procedure:
The following procedure will be used for amplifying the DNA and preparation of the PCR
fragments for sequencing on the ABI 3100.
For PCR the master mix will be prepared containing the following solutions
Reagents
Volume (µl)
10x PCR buffer
2.5
dNTPs (2.5mM)
2
Forward Primer (10 µM/µl)
0.6
Reverse Primer (10 µM /µl)
0.6
Hot Star Taq polymerase (5u/µl)
0.3
Distilled Water
17
DNA template (50ng/µl)
2
Total volume
25
4
From each diluted DNA sample 2 µl were taken and transferred to a PCR plate, and 23 µl of
the master mix (mentioned above) was dispensed in each well. The plate was centrifuged at
3000 rpm for 1 minute and placed in a PTC-200 Peltier thermal cycler-MJ research.
The PCR is performed as follows:
Initial denaturation for 15 minutes at 95ºC, when Hot Star Taq polymerase was used in the
PCR reactions intended for sequencing as better results were achieved. However, other
ordinary Taq polymerases was also used, but the initial denaturation was 5 minutes at 95ºC
followed by 34 cycles of denaturation at 95°C for 1 minute, annealing at appropriate
temperature (Table 2.1) for 1 minute and extension at 72º C for 1 minute and terminated by
extension at 72°C for 10 minutes. After completion of the 35 cycles, the PCR product
(5 µ l) was mixed with 2 µ l of orange dye and subjected to electrophoresis in 2 % agarose
gel and the remaining PCR product was stored at 4°C until required for sequencing or
restriction endonuclease digestion.
Cycling parameters:
Temperature (ºC)
95
95
At
72
72
4
Time (min)
15 (when using hot star Taq)
1
Repeat to
34 number
1
of cycles.
1
10
forever
5