Thursday, 8 April 2021

Polymerase Chain Reaction(PCR) : Basic Concept, 3 Steps, Types & Applications

  Polymerase chain reaction or PCR is an in vitro technique for generating large quantities of a specific DNA sequence. In simple words it is an automated version of DNA replication. A typical PCR reaction produces millions of copies of the amplified target DNA segment from the original DNA molecule.

  PCR technique was invented by Kary Mullis in 1985. He received Nobel Prize in Chemistry for the invention of PCR in the year 1993.

  Polymerase chain reaction the name it self tells us that in this technique DNA polymerase is used to produce copies of a target DNA sequence. It is a change reaction because the target DNA is repeatedly replicated as long as the procedure of this technique continues.

The Basic Principle behind the PCR :

   DNA double helix can be dissociated or melted by heating. This is known as denaturation. Once DNA strands are separated, it can be copied each of the two separated strands by using primers, deoxy nucleotides and DNA polymerase.

  Primer is allowed to bind to the separated strands. This is known as annealing. DNA polymerase carries out the process of DNA synthesis. This is known as extension.

  The result of this we will get duplicated DNA sequences. This process can be repeated many times and as a result we get many copies of the original DNA sequence.

Schematic representation of PCR reaction and replication of DNA

Some additional points about polymerase chain reaction

• PCR is a cyclic process.
• It consists of a series of 30 to 35 cycles.
• Each cycle has three steps :
   -  Denaturation
   -  Annealing and
   -  Extension
• Each cycle lasts from 3 to 5 minutes. This means we can have millions of copies of target DNA sequence in approximately 2.5 hours.

•  Polymerase chain reaction (PCR) takes place in small tubes made up of polypropylene. These tubes are known as PCR tubes.

• To automate PCR reaction these PCR tubes are kept in an instrument known as thermal cycler. It is also simply called as PCR machine.

• Thermal cycler is an automatic temperature control device. It automates cycling and incubation times for the reaction.
 

Requirements for PCR reaction

  PCR is an in-vitro DNA replication reaction so there are four basic components required for this reaction to take place.

1). Double-stranded DNA segment

  It is the source of the target DNA sequence that is to be copied in PCR. Both the strands of the DNA sequence act as template strands for DNA replication. One important point here is that we should have prior knowledge of the sequences at the border of the target gene segment. This is essential for our second requirement.

2). 2 Different Single-Stranded DNA primers

  Primers are short approximately 12 to 24 nucleotides long, chemically synthesized DNA sequences.

  One of these primers is complementary to the border sequence of one strand and second primer is complementary to the border sequence end of the other strand.

  Primers will bind to these DNA strands such that, their 3' prime end will point towards each other.

3). Four deoxynucleotide triphosphates (dNTPs)

(dATPs, dGTPs,dCTPs,dTTPs)
   These deoxy nucleotides will be used by DNA polymerases to synthesize new strands during the replication process.

4). Taq (DNA) polymerase

   The fourth requirement is the enzyme, which catalyze the DNA replication reaction. A heat-stable DNA polymerase. This is because PCR is carried out at higher temperatures where normal DNA polymerase enzymes will lose their structure and therefore stability and their function.

  The most often used DNA polymerase in PCR is called Taq polymerase. Taq polymerase is named after the thermophilic bacterial species Thermus aquaticus. This bacterium lives in hot springs at near boiling conditions.

Other more efficient polymerases for PCR are :
Pfu Polymerase (Pyrococcus furiosus)
Tli Polymerase (Thermococcus litoralis)
Tth Polymerase (Thermus thermophiles).

   All these reaction components are added in the PCR tube along with suitable buffer and then these tubes are kept in thermal cycler.

Steps of PCR reaction

1). Denaturation at 94-98°C

  The first step of PCR reaction is known as denaturation. In this step the reaction mixture is heated temperature during the step is about 94°C to 98°C for 45 to 50 seconds. It depends on the G+C content of DNA.

  At this temperature the double-stranded DNA in the mixture denatures into single strands.

2). Primer Annealing at 40-60°C

   The second step is known as primer annealing. sometimes also called as hybridization. In this step the temperature is slowly reduced. Temperature is about 40-60°C. At this temperature hybridization of primers to their complementary sequences on the DNA template strands takes place.

   In PCR both the separated DNA strands act as the template and we have two different DNA primers. So on first DNA template strand the primer binds complementary to the nucleotides at the border of the target sequence on the strand. Similarly other primer binds on the second DNA template strand. Note that three prime ends of both the primers are pointing towards each other.

3). Extension at 72°C

Third step is known as primer extension or simply extension. In this step the temperature is raised to about 70-75°C more specifically 72°C temperature is used. This temperature is optimum for the catalytic functioning of taq DNA polymerase.

  So in the third step Taq polymerase start extending the primer by copying the complimentary target DNA sequence. DNA synthesis is initiated at the 3' prime end of each primer and uses the separated DNA strands as a template.

  Afterer the completion of the first cycle the number of copies of the target sequence is doubled. We started with a single DNA sequence and now at the end of the first cycle, we have two copies of the DNA sequence. Here each of these two end products are made up of one original DNA strand and one newly synthesized long strand. 

Second Cycle of PCR

  Remember that for second cycle we have now two DNA double strands. That means after the denaturation step in the second cycle we will have four DNA template strands two of these are the original DNA stands and two are the newly synthesized strands.

  One of the two double-stranded DNA in the second cycle it undergoes denaturation annealing and extension again.  DNA sequence got doubled and we get four DNA copies.

  This cyclic process of DNA replication goes on doubling the DNA copies. With each subsequent cycle short template strands become more abundant. Because of this the target DNA sequence is successfully amplified.

Types of PCR

- Real-time PCR
- Quantitative real time PCR (Q-RT PCR)
- Reverse Transcriptase PCR (RT-PCR)
- Multiplex PCR
- Nested PCR
- Long-range PCR
- Single-cell PCR
- Fast-cycling PCR
- Methylation-specific PCR (MSP)
- Hot start PCR
- High-fidelity PCR
- In situ PCR
- Variable Number of Tandem
- Repeats (VNTR) PCR
- Asymmetric PCR
- Repetitive sequence-based PCR
- Overlap extension PCR
- Assemble PCR
- Intersequence-specific PCR(ISSR)
- Ligation-mediated PCR
- Methylation –specifin PCR
- Miniprimer PCR
- Solid phase PCR
- Touch down PCR, etc

Application of PCR :

PCR has limitless applications some important application are as of
- PCR include evolutionary studies - Forensic analysis such as for paternity testing, crime investigation
- Genome sequencing projects
- Disease diagnosis
- Agricultural testing etc.

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