Polymerase Chain Reaction (PCR):  Steps, Types, Applications

PCR Cycle and Target copies

Polymerase chain reaction (PCR) is an efficient and cost-effective molecular tool to copy or amplify small segments of DNA or RNA.  PCR combines the principles of complementary nucleic acid hybridization with those of nucleic acid replication that are applied repeatedly through numerous cycles. It results in the exponential production of the specific target DNA/RNA sequences by a factor of 10^7 within a relatively short period.

This in vitro amplification technique can amplify a single copy of nucleic acid target by using two synthetic oligonucleotides “primers” that bind to the target genomic sequence, which are extended by a  Taq polymerase (a thermostable DNA polymerase). An automated process of repeated cycles (usually 25 to 40) of denaturation of the template DNA (at 94°C), annealing of primers to their complementary sequences  (50°C), and primer extension (70°C) are employed for the amplification of target sequence.

PCR was originally developed in 1983 by the American biochemist and Nobel Laureate Kary Mullis.

Primer:  A short segment of nucleotides, which is complementary to a section of the DNA or RNA, which is to be amplified in the PCR. Two short DNA sequences designed to bind to the start (forward primer) and end (reverse primer) of the target sequence is used in PCR.

Taq polymerase: A thermally stable DNA polymerase originally isolated from the thermophilic bacterium Thermus aquaticus, which resist inactivation during denaturation temperatures and allows primer extension at high temperature.

Components of Polymerase Chain Reactions (PCR)

I was excited and pose for photo (few years back), when our Lab got the Real-Time PCR first time.
I was too excited when our lab got the Real-Time PCR first time (File photo).
  • DNA template (the sample DNA that contains the target sequence to amplify)
  • Deoxyribonucleoside triphosphates (dNTPs)
  • PCR buffer
  • Primers (forward and reverse)
  • Taq polymerase

 

Steps of polymerase chain reaction-PCR

To perform PCR, the extracted sample (which contains target DNA template) is added to a tube containing primers, free nucleotides (dNTPs), and Taq polymerase. The PCR mixture is placed in a PCR machine. PCR machine increases and decreases the temperature of the PCR mixture in automatic, programmed steps which generates copies of the target sequence exponentially.

Polymerase Chain Reaction (PCR) has three major steps.

Steps of Polymerase Chain Reaction
Steps of Polymerase Chain Reactions (PCR)
  1. Denaturation (strand separation): The separation of the two hydrogen-bonded complementary chains of DNA into a pair of single-stranded polynucleotide molecules by a process of heating (94°C to 96°C)
  2. Annealing (primer binding): The temperature is lowered (45-60 °C) so the primers can attach themselves to the single-stranded DNA strands.
  3. Extension (synthesis of new DNA): It starts at the annealed primer and works its way along the DNA strand (72°C).

Once the first round is completed, the process is repeated by cycling back to the first reaction temperature and the next round of denaturation, annealing, and extension is started (an automatic process in thermocycler). This 3 steps temperature cycle is repeated approximately 30 times which results in exponential amplification of the target gene sequence.

Animation

Detection of PCR products

Labeled probe that is specific to the target gene sequence is used to detect PCR amplified gene product (also known as amplicon).  Based on the nature of the reporter molecule used, the probe generates radioactive, colorimetric, fluorometric, or chemiluminescent signals. Probe-based detection of amplicons serves two purposes

  1. It allows visualization of the PCR product
  2. It provides specificity by ensuring that the amplicon is the target sequence of interest and not the result of non-specific amplification.

Apart from DNA based hybridization method, sometimes a simple gel electrophoresis method is sufficient to confirm the presence of specific amplicons.

Types of polymerase chain reaction-PCR

Several modifications of PCR methods have been developed to enhance the utility of this method in diagnostic settings based on their applications. Some of the common types of PCR are;

  1. Real-Time PCR (quantitative PCR or qPCR)
  2. Reverse-Transcriptase (RT-PCR)
  3. Multiplex PCR
  4. Nested PCR
  5. High-fidelity PCR
  6. Fast PCR
  7. Hot-start PCR
  8. GC-rich PCR
  9. Long-range PCR
  10. Arbitrarily primed polymerase chain reaction (AP-PCR)

Applications of PCR

  • Identification and characterization of infectious agents
    • Direct detection of microorganisms in patient specimens
    • Identification of microorganisms grown in culture
    • Detection of antimicrobial resistance
    • Investigation of strain relatedness of a pathogen of interest
  • Genetic fingerprinting (forensic application/paternity testing)
  • Detection of mutation (investigation of genetic diseases)
  • Cloning genes
  • PCR sequencing

References & further readings:

  • Animation source: dnalc.org
  • Image source: Wikipedia.org 
  • Bailey& Scott’s Diagnostic Microbiology- 12th Edition

Acharya Tankeshwar

Hello, thank you for visiting my blog. I am Tankeshwar Acharya. Blogging is my passion. As an asst. professor, I am teaching microbiology and immunology to medical and nursing students at PAHS, Nepal. I have been working as a microbiologist at Patan hospital for more than 10 years.

10 thoughts on “Polymerase Chain Reaction (PCR):  Steps, Types, Applications

  1. Hello Mr. Tankeshwar, I need some insight on different types of PCR and qPCR. Please give me your email id so that mail you the details

    1. Informative article… here is another article which i think you must have a look to know more about PCR.
      here is the link: https://www.scienceofhealthy.com/polymerase-chain-reaction-pcr/
      hope you’ll find it useful…

  2. Mr. Acharya
    This is a great article on PCR. It covers also every aspect of it.
    These are few tips about Improving PCR results, If you link it from your article, it will help your readers to learn here
    http://techooid.com/better-pcr-results

    Please keep writing 🙂

  3. i read this page completely,
    there is so wonderful information for me ,
    i’m new student of molecular biology.
    that’s why very little quarries should be note for my exam preparation.
    there is a little mistake in this page ,
    i’m doing to point out & kindly correct it.
    (the purpose of point out is only correction)
    PCR cycle 30
    and the target copies are 1073741824 not 1073741842.

  4. I didn’t found it so worth, because all this information found easily in any lab protocol, better u have focused on types of PCR and elaborated it, which will helpful.

  5. Thanks this post is very very useful for me means for my project in biology.
    I appreciate u for posting this post and I am again thanking you .

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