Lecture03
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
appropriate primers
can order
2. Heat stable polymerase.
3. Four d NTPs.
4. Thermocycler (standard, but optional)
Changes temperature very rapidly for each cycle. (denature, anneal, extend).
Lecture03
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 triphosphate), 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 DNAsequence 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
Taq-polymearase. 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 20-25 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.
Lecture03
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
DNA-matrix, 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 cycles that, there is enough DNA to be detected and analyzed.
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 DNA-recognition-sequence (according to their complementary
bases) of the single-stranded DNA- template-sequence.