Polymerase Chain Reaction (PCR)

PCR stands for Polymerase Chain Reaction, which is a laboratory technique used to make multiple copies of a segment of DNA. It is simple, rapid, and accurate. It is an inexpensive tool used to copy or amplify, a specific DNA target from a mixture of DNA molecules. In 1983, this technique was developed by Kary B. Mullis. In 1993, Mullis along with biochemist Michael Smith were jointly awarded the Nobel Prize in Chemistry (in appreciation of their contribution to science). This technique has a large range of applications like gene cloning, sequencing, forensic sciences, and diagnosis of different types of diseases in biological research.

Also check out- Salient features of a cloning vector – My Biology Dictionary

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DISCOVERY OF POLYMERASE CHAIN REACTION (PCR):

In 1983, Mullis was working as a chemist for one of the biotechnology companies, Cetus Corporation. He had an idea that using DNA polymerase a pair of primers can be used to copy the DNA sequence. On December 16, 1983, Mullis completed demonstrating Polymerase Chain Reaction. Other scientists, Randall Saiki and Henry Erlich of the same Corporation also worked on the PCR project. They tried to test whether PCR could amplify a specific human gene (Betaglobin) from genomic DNA. They published their first paper on the utilization of the technique.    

            Consequently, Mullis was still working on a paper that would describe PCR itself. The polymerase he used for the amplification was broken or destroyed at high temperatures. In 1986, Saiki started to use Taq Polymerase (Thermophilus aquaticus) DNA polymerase to amplify segments of DNA. It acts heat resistant so only needs to add once. Thus, making this technique more affordable and subjected to automation. This technique created a revolution in genetics, molecular biology, biochemistry, medicines, and forensics.

PRINCIPLE OF POLYMERASE CHAIN REACTION (PCR)

The basic principle of the PCR techniques is the enzymatic replication of DNA. With this technique, short a segment of DNA is amplified using primer-mediated enzymes. Then, DNA Polymerase synthesized new strands of DNA complementary to the template DNA. After that DNA polymerase adds a nucleotide to the pre-existing 3’-OH group only. Thus, DNA replication always starts with the primer. More nucleotides are joined to the 3’ prime end of the DNA polymerase. Most PCR techniques amplify DNA fragments of between 0.1 and 10 Kilobase pairs (kbp) in length. Some techniques allow for the amplification of fragments up to 40 kbp.

 COMPONENTS OF THE POLYMERASE CHAIN REACTION (PCR):

For a basic PCR set-up, several components and reagents are required like DNA templates, primers, DNA polymerases, nucleotides, and buffers.

TEMPLATE:

The DNA template is the DNA of interest from the sample. To amplifies the target DNA sequence, whole genomic DNA or plasmid DNA can be used as a template for carrying out PCR. It contains the DNA target region to amplify.

PRIMERS:

Primer is the small pieces of single-stranded DNA that are complementary to the target sequence. They are synthesized artificially in laboratories. They are synthesized in pairs- a forward primer and a reverse primer. A forward primer is a compliment of the same sequence towards the other end. A reverse primer is the reverse complement of the same sequence towards the other end. To synthesize new DNA, one end of separated single-stranded DNA binds to the forward primer and the other end of the complementary strand binds to the reverse primer through complementary binding.

Properties of primers:

• The length of the primers is usually 18 to 25 bases.
• It has a definite melting temperature between 60°C and 65°C.
• Primers above 65°C tend to secondary annealing.
• Primers should not be self-complementary.
• The content of bases G or C together should be 40% to 60%.
• To promote binding and specificity, the 3’ of a primer should be C or G.

DNA POLYMERASE :

The new strand of DNA complementary to the target DNA is synthesized by an enzyme called DNA polymerase. Taq polymerase is the mostly used enzyme which is isolated from the heat-resistant bacteria Thermus aquaticus. It is thermostable. There is no change in structure at very high temperatures for the synthesis of target DNA. There are various other polymerases apart from Taq DNA polymerase. For example, Pfu polymerase isolated from the single-celled archaeon Pyrococcus furiosus, Vent polymerase isolated from Thermococcus litoralis etc.

dNTPs (deoxynucleotide triphosphates):

dNTPs provide energy for polymerization. These are the building blocks for the synthesis of DNA. It is a single unit of bases. It joined the nucleotide bases floating in the reaction mixture for synthesis.

BUFFER:

Buffer is a suitable medium for any enzymatic reaction in which the enzymes can function. It provides optimal pH and optimum concentration to stabilize the reaction.

DIVALENT CATIONS:

All the thermostable DNA polymerases for their activity need free divalent cations. Moreover, for the optimum temperature, Mg++ ions are usually used.

THERMAL CYCLER:

A thermal cycler is a machine that is carried out in a PCR. It is a process of heating and cooling that creates the conditions necessary for DNA replication. It works on the principle of the Peltier effect. 

PROCEDURE OF POLYMERASE CHAIN REACTION (PCR):

The PCR involves three major steps-

• Denaturation
• Annealing
• Extension

All the above three steps are repeated in 30-40 cycles to amplify the target DNA in large quantities.

DENATURATION:

In the first step, when the reaction mixture is heated to 94–95℃ for about 0.5 to 2 minutes then Denaturation occurs. It breaks the hydrogen bonds between the two strands of DNA. Thus, converts it into single-stranded DNA.

For the production of new strands of DNA, the single-stranded DNA act as a template. To ensure the separation of the two strands temperature should be provided for a longer duration.

ANNEALING:

In the second step, nearly 50-65℃ temperature is provided for 35-40 seconds. Then, the primers can anneal to the single-stranded DNA. Here, the primers joined to their complementary sequences on the template DNA.

Primers are single-strand sequences of DNA or RNA around 20-30 bases in length. It serves as the starting point for the synthesis of DNA.

ELONGATION:

In the third step, 72℃ temperature is provided for 7-9 minutes. The bases are joined to the 3’ end of the primer by the Taq polymerase enzyme. It elongates the DNA in the 5’ to 3’ direction. At optimum conditions, DNA polymerase adds about 1000bp/minute.

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TYPES OF POLYMERASE CHAIN REACTION (PCR):

These are the different types of PCR-

REAL-TIME PCR:

With the help of a fluorescent reporter, the DNA amplification is detected in real-time. The signal strength of the fluorescent is directly proportional to the number of amplified DNA molecules.

NESTED PCR:

It reduces the non-specific binding of products due to the amplification of unexpected primer binding sites.

MULTIPLEX PCR:

It is used for the amplification of multiple targets in a single PCR experiment. It produces many copies of different DNA sequences.

QUANTITIVE PCR:

This is used as the DNA amplification linearity to detect, characterize and quantify a known sequence in a sample.

ARBITRARY PRIMED PCR:

This is used as DNA fingerprinting technique based on PCR. It is used as a primer the DNA sequence of which is chosen arbitrarily.

APPLICATION OF POLYMERASE CHAIN REACTION (PCR):

Medicine:

• For monitoring the gene in gene therapy
• For Detecting disease-causing genes in the parents
• For Testing for genetic disease mutation

Forensic science:

• For tools in genetic fingerprinting
• For identifying the criminal from millions of people
• For Paternity tests

Research and genetics:

• For Gene Mapping
• For analysis of gene expression
• To compare the genome of two organisms in genomic studies

To sum it up, the discovery of the polymerase chain reaction brought huge benefits and scientific developments such as gene expressions, and forensic science and is also used for the diagnosis of infectious diseases. Currently, the appliances required to undertake PCR are bulky and expensive, but some new miniature devices are being developed to allow the benefits of PCR to be taken out of the laboratory.

Team MBD