Real time PCR
The real time polymerase chain reaction is also
called as q-PCR (quantitative PCR).
It is a laboratory technique based on PCR,
which is used to amplify and simultaneously quantify the targeted DNA molecule.
Its key feature is that the amplified DNA is
detected as the reaction proceeds in real time.
The common methods:
1.
Use
of non-specific dyes: the nonspecific dyes are not complementary to any
specific sequences of the DNA, rather it intercalates with the double stranded
DNA.
2.
Sequence
specific fluorescent probes: consists of oligonucleotides that are labeled with
fluorescent reporters, which permits detection only after hybridization of
probe with targeted DNA.
3.
The
probe is tagged with fluorescent materials (reporter) along with is attached
the quencher molecule in very close proximity with the reporter. The quencher
quenches (absorbs) the fluorescence of the reporter.
4.
The
probe is first hybridized with the DNA, then the polymerase is allowed to
perform its task, as the DNA polymerase adds nucleotides to the template
strand, the flurophore is released away from the quencher molecule and the
fluorescence is produced, depicting the formation of double stranded DNA. The
intensity of fluorescence is directly proportional to the quantity of the ds
DNA formed.
