Polymerase chain reaction (PCR) is a major technique which is used to analyze the DNA with high accuracy. It can be defined as “Fast, simple and inexpensive way to amplify (copy) small quantities of specific DNA fragments via different polymerase enzymes by using in-vitro methods”. The product of PCR is commonly known as “amplicon”.
Why May You Want to Amplify DNA?
1. To synthesize copies of the gene which is infrequent or rare.
2. To check the relative abundance of two DNA samples.
How PCR Revolutionize Molecular Biology?
It was very difficult to analyze DNA in past but now it has become very easy due to polymerase chain reaction. PCR revolutionize the scientific approaches by its versatile applications in the research field. Karry Mullis developed PCR in 1983, who was the winner of the noble prize in chemistry in 1993. The Polymerase chain reaction is an easy, reliable and inexpensive process to repetitively replicate a segment of DNA, in molecular biology, the most widely used technique is PCR. Now PCR is a necessary technique which is used in research labs and in diagnostic labs. In 30 cycles of PCR millions of copies of template DNA fragment {230 – (2 x 30)} can be produced.
Components of PCR
dNTPs
dNTPs
(deoxynucleotide triphosphate) are the
major component of PCR. Polymerase enzyme adds dNTPs to the growing strand in
the reaction after removing two phosphates.
Polymerase enzyme
The polymerase enzyme that is used in PCR should be thermally stable like Taq polymerase which can perform their function easily at 95°C and do not denature.
For example, Taq polymerase, Pfu polymerase etc.
MgCl2
MgCl2
helps in the enzymatic activity of polymerases for the addition of dNTPs to the
strand. It also promotes hybridization of the primer to the template (denatured
strand) at the target region. DNA
molecules have a negative charge on it so due to repulsion between template strands
and primers hybridization become difficult. Mg2+ ions embrace these
strands and minimize the repulsion effect by neutralizing the net charge on the
strands.
Its
concentration is important to get a high
yield of the final product. Due to the high
concentration of MgCl2, non-specific binding of primers will increase.
Low concentration reduces the primer binding.
DNA Template
This
is the target DNA and contains the gene
of interest which is to be amplified. The sequence of the target region of the template strand must be
known.
The fragment of DNA to be amplified should be less than 3 kb in size and for ideal condition less than 1kb. By standard PCR techniques, fragments of 10 kb can be amplified but by increasing the length of the fragment amplification process will become less efficient. By using special methods amplification of larger fragments (up to 40 kb) is possible.
Primers
Primer is a short fragment of DNA that serves as an initiating point for DNA synthesis. Forward and reverse primers are used in a polymerase chain reaction. Both of these primers hybridize with each strand of template DNA at 5′ end in such a way that both primers have 3′ end towards each other. The size of primers that can be used for the polymerase chain reaction typically 20-30 bases in length.
Size
of primer should be normal otherwise non-specific
binding increases by using too short primers. Short primers hybridize easily
with the sites other than the specific complementary region on target DNA. By using
too large primer hybridization time increases due to this final yield of the
PCR in a specific time decreases.
Buffer
Buffer solution maintains the pH of the reaction mixture which is important for proper functioning and stability of DNA polymerase.
How PCR Works?
The
machine which is used for the polymerase chain reaction is known as a thermal cycler, the programmed machine that can
perform an automated function has become the basic need for the
polymerase chain reaction in these days.
Initiation/ Denaturation
This is the first step in the polymerase chain reaction. In PCR reaction template strand has double-stranded structure so to amplify the gene of interest it is necessary to melt the double-stranded structure. 92 °C to 94 °C for 1 minute is required to break the hydrogen bonding between the nitrogenous bases of the target DNA and denature the double-stranded structure.
Annealing
The hybridization process of the primers to the target DNA is called annealing. Low temperature is required for the annealing process for 1minute. At 50-60°C some single strands of the template rejoin with each other but also primers bind easily with template strand at their complementary region.
It is important to note that annealing temperature should be less than Tm (melting temperature) of the primers, otherwise, primers will not anneal. GC content of the primer plays a major role, primer with high GC content has high melting temperature and primer with low GC content usually has a low melting temperature. Tm for the primers must be calculated before the reaction.
Elongation
After
the annealing of primer to the template elongation starts. Taq Polymerase adds
nitrogenous bases to the 3′ end of the primer at high temperature 74°C (for 2
minutes) just below to the optimum temperature of the Taq polymerase.
A
set of “long product” is synthesized in this first stage of the PCR from each
strand of the template target DNA. The product of this first stage PCR reaction
has identical 5′ end but different 3′ end
because reaction starts from the same site where primer gets attached but ends
at different sites where reaction stops by chance.
Final Elongation
Final elongation is an optional step to make sure that any remaining single-stranded DNA is fully elongated. This elongation needs 70-74°C for 5 to 15 minutes after the last cycle of PCR. Final elongation is also helpful in the addition of 3′ overhang by polymerase enzyme that is used in TA cloning.
Final Hold
At the end, the final hold is maintained at 4-15°C for an indeterminate time.
PCR Optimization:
How temperature affects the PCR?
the primer should be very specific to the sequence of interest and the role of annealing temperature is important for specific hybridization because DNA-DNA hybridization is a temperature-dependent process. Hybridization of primers to the target region will not occur at high temperature hence primer and template remain dissociated. At low-temperature mismatch hybridization will be there (not all the base pairs correctly formed). So, amplification will occur at a non-target site in the template DNA molecule.
The ideal temperature for the hybridization of primers is 1-2°C less than melting temperature (Tm) of the primer-template hybrid. This temperature allows hybridization of the primer to target region but mismatch pairs will not form. Hence the melting temperature of a primer-template hybrid must be known for better results. This formula is very helpful to find the melting temperature of the primer-template hybrid:
Tm= (4 × [G + C]) + (2 × [A + T]) °C
In
this [A + T] is the number of Adenine, thiamine base pairs in the sequence of
primer and [G + C] is the number of guanine, cytosine base pairs in the primer sequence. It is
important to note that melting temperature should be identical for both reverse
primer and forward primer, otherwise one of these primers will not accurately
bind.
Primer design
During primer designing the following points are important.
GC content: Melting temperature directly depends upon the GC content in the primer-template hybrid because three hydrogen bonds form between G and C nitrogenous bases in DNA double-stranded structure. Greater the GC content higher will be the Tm. So, primers should have 40-60% GC content normally.
GC clamp: within the last five bases at 3′ ends of the primers should have G or C bases that promote specific binding at the 3′ ends due to strong bonding. More than three G or C repeats should be avoided.
Due to intramolecular or intermolecular interactions sometimes, self-secondary structures are made which can lead to poor yield because they greatly decrease the availability of the primers to the reaction like
Self-dimer: sometimes by intermolecular interactions same(sense) strand hybridize with each other and form self-dimers.
Cross dimes: by intermolecular interactions between sense and antisense primers cross dimers can form.
Hairpins: due to intramolecular interaction in the primer hairpin is formed.
Buffers
Most of the time in different
concentrations of KCl and tris are used as a buffering agent for the polymerase
chain reaction. KCl helps in primer binding but its high concentration may
inhibit the activity of the polymerase enzyme. DMSO, glycerol, and BSA also used.
After the Polymerase Chain Reaction (PCR): Studying Product
The product after polymerase chain
reaction can be analyzed by three different
techniques.
Gel electrophoresis of PCR products
Cloning of polymerase chain reaction products
Sequencing of polymerase chain reaction products.
you want to study GEL ELECTROPHORESIS in detail? click here.
Types of PCR
Colony polymerase chain reaction
Nested polymerase chain reaction
Multiplex polymerase chain reaction
AFLP polymerase chain reaction
Hot Start polymerase chain reaction
Long polymerase chain reaction
Real-time polymerase chain reaction
Applications of Polymerase Chain Reaction (PCR):
Molecular Identification:
Molecular archaeology
Detection of pathogens
Drug discovery
Mutation screening
Genotyping
Classification of organisms
You want to know More about DNA fingerprinting and Genetic matching ?
Sequencing:
Bioinformatics
Human genome project
Genome cloning
Genetic Engineering:
Site-directed mutagenesis
Gene expression studies