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Published: 03/02/2023|Last Updated: 14/11/2023
PCR or Polymerase Chain Reaction is the process used in Molecular Biology
for the amplification of genomic DNA samples when the sample count is very
less. It produces millions of samples. This technique was developed by
Kary Mullis. It is the application of Taq polymerase which is a heat-stable DNA Polymerase. This can synthesize a new strand of DNA called the daughter strand.
for the amplification of genomic DNA samples when the sample count is very
less. It produces millions of samples. This technique was developed by
Kary Mullis. It is the application of Taq polymerase which is a heat-stable DNA Polymerase. This can synthesize a new strand of DNA called the daughter strand.
Read more about Molecular Biology on Introduction to Molecular Biology
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Key components for a PCR
- Template DNA
- Primer for 3′-OH end
- Four dNTP’s
- Taq polymerase
In Detail
1. Template DNA
It is a DNA that contains the target sequence. Initially, it is heated at a high temperature to unwind into a single strand. This process is called denaturation.
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2. Primers
These are the short sequence containing portions which can be complementary to the target sequence of the DNA. Taq polymerase helps in the formation of the new strand from the primer. Two primers for each 3′-OH end are used.
3. Deoxynucleotides or dNTPs
It contains a combination of bases and phosphate group, the bases can be Adenine, Thymine, Guanine & Cytosine. That is A, T, G, and C.
- dATP – deoxyadenosine triphosphate
- dGTP – deoxyguanosine triphosphate
- dCTP – deoxycytidine triphosphate
- dTTP – deoxythymidine triphosphate
4. Taq polymerase
Taq polymerase is a DNA polymerase that can withstand high temperatures during denaturation as well as annealing. It is obtained from Thermus aquaticus. It can generate new DNA strands.
Major Steps of PCR
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1. Sample Preparation
It is the process that involves the extraction of the target DNA sequence using the chemical or physical method. Then all the materials added during the process are loaded along with the sample in a vial.
2. Denaturation of DNA (Separation)
During this step, the temperature of the system got risen to 95 degrees Celsius, which separates the double-stranded DNA into two separate strands. The hydrogen bonds between the strands got broken by the high temperature.
3. Annealing – Binding of primers to complementary sequence
It is the process in which the two Primers will bind to the complementary sequence present on the 3′-OH end of each strand. Annealing will be successful if the Primer exactly matches the target sequence. The temperature is maintained at 40 – 42 degrees Celsius.
4. Primer Extension
It is the stage in which a new copy of the DNA strand is made. The temperature is raised and managed at 74 degrees Celsius. This process takes place with the help of a heat-stable DNA polymerase enzyme called Taq Polymerase. This enzyme adds nucleotides to annealed primer. It will create a new DNA strand.
After every four stages of the PCR, one cycle is completed. Newly formed DNA can be amplified as a network. Usually, 20 – 30 cycles are conducted in a normal PCR system. This process is done in a specialized machine called thermal cycler.
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Published: 03/02/23, 0942
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[…] The amplified fragments can be observed using autoradiography or fluorescence techniques on denaturing polyacrylamide gels. AFLPs are DNA fragments (80–500 bp) produced by restriction enzyme digestion, oligonucleotide adapter ligation to the digestion products, and targeted PCR amplification. […]