REAL TIME PCR, principle of real time pcr, method for detection real time pcr, taq man probe, molecular beacons. application of real time pcr. difference between real time pcr and conventional pcr.

1. SUBJECT: MEDICALBIOTECHNOLOGY
SUBJECT CODE: MCE 804
SUBMITTED TO:
DR. RAJIV KANT
JIBB
SUBMITTED BY:
GAURAV AUGUSTINE
ID: 19MTBT001
M.TECH BIOTECH
2ND SEM

2.  A real-time PCR monitors the amplification of a
targeted DNA molecule during the PCR. It is also
known as Quantitative polymerase chain reaction
(q PCR).
 The important feature of Real-Time PCR is
amplification during each PCR cycle is monitored
in a real-time manner.

3.  The two method for the detection of product in
Real-time PCR:
1. Non-specific fluorescent dyes that intercalate
with any double-stranded DNA.
2. Sequence-specific DNA probes consisting of
oligonucleotides that are labelled with a
fluorescent reporter, which permits detection
only after hybridization of the probe with its
complementary sequence.

4.  In real time PCR, we can calculate or measure the
amount of the amplification which is not possible
in the conventional PCR.
 In the conventional PCR, required to do agarose
gel electrophoresis that is also not required in case
of the real-time PCR. By doing the melting curve
analysis one can get results.
 In real time PCR amount of gene expression can
be measured.

5.  The principle of real-time PCR relies on the use of
fluorescent dye.
 Real-time PCR is carried out in a thermal cycler
with the capacity to illuminate each sample with a
beam of light of at least one specified wavelength
and detect the fluorescence emitted by the excited
fluorophore.
 The thermal cycler is also able to rapidly heat and
chill samples, thereby taking advantage of the
physicochemical properties of the nucleic acids
and DNA polymerase.

7.  In PCR, DNA binding dyes binds to all dsDNA
which increasing the fluorescence quantum yields
of the dye.
 An increase in DNA product during PCR therefore
leads to an increase in fluorescence intensity
measured at each cycle.
 However, dsDNA dyes such as SYBR Green will
bind to all dsDNA PCR products, including
nonspecific PCR products such as primer. This can
potentially interfere with, or prevent, accurate
monitoring of the intended target sequence.

8. •In Real-time PCR, the reaction is prepared as usual with
the addition of fluorescent dsDNA dye.
• Then the reaction is run in a real-time PCR instrument,
and after each cycle, the intensity of fluorescence is
measured with a detector; the dye only fluoresces when
bound to the dsDNA. SYBR green and EvaGreen are two
main dye used in the quantitative real-time PCR. The
experiments are used in the validation of the assays such as
DNA microarray.

9.  The method used the single short sequence-
specific probes which are of two types:
1. Linear probe
2. Molecular becons
 Linear probe: Linear probes are the TaqMan
probe, relies on the activity of Taq DNA
polymerase. TaqMan probes are hydrolysis
probes that are designed to increase the
specificity of quantitative PCR.

10.  The TaqMan probe principle relies on the 5´–3´
exonuclease activity of Taq polymerase to cleave a
dual-labeled probe during hybridization to the
complementary target sequence and fluorophore-based
detection.
 The probes are the labelled short single-stranded
sequence-specific DNA molecules which are radio or
fluorescent labelled.
 Here the probe is labelled with the fluorescent dye
called a reporter molecule, at the 3’ end. The other 5’
end has the quencher dye which is in the close
proximity to the reporter dye and quenches the
fluorescence of the reporter dye.

11. •The Taq DNA polymerase used in the real-time PCR has the 5’ to 3’ exonuclease
activity, which removes the probe by extending the DNA. Once the probe is
dissociated the reported molecules emitted fluorescent. If the DNA is amplified,
the reported molecule unquenched and releases the fluorescence.
•TAMRA
and Black
Hole
Quencher
are two
widely used
quencher
dyes. While
FAM is the
most
popular
reporter
dye

12.  Molecular beacons are sensitive, structured probes:
used for sequence-specific detection. Molecular
beacons are hairpin-shaped molecules with an
internally quenched fluorophore whose
fluorescence is restored when they bind to a target
nucleic acid sequence.

13.  Beacons are the hairpin loop-like structure of the
oligonucleotides which has complementary sequences
on both the ends.
 The central loop is complementary to the target
sequences.
 One end of the hairpin loop has the quencher dye and
one end has the reporter fluorescent dye.
 when the two ends of the hairpin stem are in close
proximity with each other, the reporter molecule is
quenched and cannot generate fluorescence.
 But when it binds to the complementary sequence,
the two ends of the hairpin separated with each
other, the quencher is blocked, the reported dye
released and emits the fluorescence. The emission is
recorded by the detector.

14. •The molecular beacon probes are highly sequenced specific and
are the best choice for sensitive reactions.
•The structure of molecular beacon is very important,
Suppose, if the structure of the hairpin loop is too stable, it
cannot be separated and unquenching cannot happen.

16. •Disease diagnosis: It is used to quantify the mutated gene in the
disease patient. The quantitative real-time PCR is even used in the
determination of copy number variation in different tissues for
different inherited disorders.
•MicroRNA analysis: The qPCR assay used to quantify the
microRNA from different tissues.
•Cancer detection.
•GMOs detection: The mRNA expression quantification through
real-time PCR helps to detect the amount of the gene expressed
into the GMO.
•Genotyping and quantification of pathogens: By quantifying
the DNA from the infection sample, through the melting curve and
dissociation curve analysis the exact number of the pathogen can be
determined. qPCR is applicable in Identification, characterization,
genotyping and quantification of an infectious pathogen.
•Apart from these applications, the real-time PCR is also used in
the forensic studies, evolutionary studies, mutation creation, fossil
studies and in other applied fields.

17. The quantitative real-time PCR is an accurate, fast,
sensitive, and adequate method in the genomic
research. The diagnostic value of real-time PCR is
more than any other PCR techniques. Real-time
PCR is one the best method for quantification in
recent days.

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