Polymerase Chain Reaction

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POLYMERASE CHAIN REACTION
Polymerase chain reaction (PCR) is an in vitro method that
can be used for rapid production (amplification) of very
large amounts of specific segments of DNA. It is
particularly used for amplifying DNA for clinical or forensic
testing procedures. By PCR DNA can be amplified from a
single strand of hair or single drop of blood or semen.

Events of the PCR


Millions Of copies Of the target sequences can be readily
obtained by PCR if the flanking sequences of the target
are known. A PCR is carried Out by adding the following
components to a solution contacting DNA sequences Of
interest :

  1. A pair Of “primers” (synthetic oligonucleotide of 20 to
    35 sequences) that bind with the flanking sequences
    Of the target. One primer is complementary to flanking
    sequences in one strand of the DNA to be amplified and
    the other is complementary to flanking sequences in
    other DNA strand).
  2. All four deoxyribonucleoside triphosphates (dNTPs).
  3. A heat stable DNA polymerase (this polymerase is isolated from thermus aquaticus, a bacterium that grows in hot springs)


A PCR Cycle Consists of Three Steps:

  1. Strand separation: The two strands Of the parent DNA
    are separated by heating the solution to 95°C for 15s.
  2. Binding of primers: The solution is then cooled to
    540C to allow each primer to bind to a separated DNA
    strand. One primer binds to the 3′ end of the target on
    one strand, and other primer binds to the 3′ end on the
    complementary target strand.
  3. DNA synthesis: The solution is then heated to 72°C
    (the optimal temperature for heat stable polymerases).
    Deoxyribonucleotide triphosphates and heat stable
    DNA polymerase are added to the mixture to initiate the
    synthesis. One such DNA polymerase enzyme is Tag
    polymerase is obtained from thermophilic bacterium,
    Thermus aquatics. The polymerase elongates both
    primers in 5′ to 3′ direction.

These three steps, strand separation, binding of primers, and
DNA synthesis constitute one cycle of the PCR amplification and
can carried out repetitively just by changing the temperature of
the reaction mixture. The thermostability of polymerase makes
it possible to carry out PCR in a closed container. No reagents are
added after the first cycle. At the completion of the second cycle,
four duplexes containing the targeting sequence have been
generated. Subsequent cycles will amplify the target sequence
exponentially. Ideally, after n cycles, the desired sequence is
amplified 2n-fold. The amplification is million fold after 20 cycles
and billion fold after 30 cycles, which can be carried out in less
than one hour.

Applications of PCR

Polymerase chain reaction is a very powerful technique in medical diagnostics, forensics, and studies of molecular evolution.


Diagnostic Importance


PCR can provide valuable diagnostic information in medicine. Bacteria and viruses can be readily detected with the use of specific primers. For example,

  • PCR can reveal the presence of small amounts of DNA
    from the human immunodeficiency virus (HIV) in persons who have not yet involved an immune response to this pathogen. In these patients, conventional assays designed to detect antibodies against the virus would yield a false negative test result.
  • Finding Mycobacterium tuberculosis bacilli in tissue specimens is slow and laborious. With PCR, as few as 10 tubercle bacilli per million human cells can be readily detected.
  • PCR is promising method for early detection of cancers. PCR can identify mutations of certain growth control genes, such as the ras genes.
  • The capacity to greatly amplify selected regions of DNA can also be highly informative in monitoring cancer chemotherapy.
  • Tests using PCR can detect when cancerous cells have been eliminated and treatment can be stopped; they can also detect a relapse and the need to immediately resume treatment.
  • PCR is ideal for detecting leukemias caused by chromosomal rearrangements.
  • In clinical biochemistry and molecular biology it is used in the antenatal diagnosis of single gene mutation and in studying structural gene polymorphism.

Forensics and Legal Medicine

  1. PCR is also having an effect in Forensics and legal medicine.
    The root of a single shed hair found at a crime scene contains
    enough DNA for capturing by PCR.
  2. PCR amplification of multiple genes is being used to
    establish biological parentage in disputed paternity and
    immigration cases.
  3. Analysis ofblood stains and semen samples by PCR have
    implicated blame or innocence in numerous assault
    and rape cases.

Studies of Molecular Evolution

  1. PCR provides an ideal method for amplifying ancient DNA
    molecules so that they can be detected and characterized.
  2. PCR can also be used to amplify DNA from micro-
    organisms that have not yet been isolated and cultured.
  3. The sequences from these PCR products can be sources
    of significant awareness into evolutionary relationships
    between organisms.