PCR Variant Flashcards
a process that
involves a reverse transcriptase
(RTase),an enzyme that uses RNA as the
template to make complementary DNA
(cDNA)
- This process is the exact opposite of the
naturally occurring DNA transcription in
which RNA is synthesized using DNA as
the template
Reverse transcription
used to amplify multiple target sequences in a
single PCR reaction using multiple primer
sets.
- This method is used to detect deletions,
polymorphisms, mutations, etc.,
-also used to detect
different viral, bacterial and other pathogens in a single tube.
- This method consumes less time and
effort in obtaining the results
Multiplex PCR
- a modification of PCR that
was designed to improve sensitivity and
specificity. - involves the use of two primer
sets and two successive PCR reactions. External primers and Nested primers.
Nested PCR
- detection of Rickettsia, Bartonella, and similar organisms in blood
(bacteremia) and tissues, - detection of herpesvirus and enterovirus in the CSF
- detection of M. tuberculosis in sputum sample
Application of Nested PCR
It monitors the amplification of a targeted
DNA molecule during the PCR (i.e., in real
time)
qPCR
Two common methods for the detection of
PCR products in real-time PCR are:
1)non-specific fluorescent dyes
(2) sequence-specific DNA probes consisting
of oligonucleotides
intercalate with any double-stranded DNA
1)non-specific fluorescent dyes
are labelled with a
fluorescent reporter, which permits detection
only after hybridization of the probe with its
complementary sequence
sequence-specific DNA probes
The_____ of the real-time PCR reaction refers to the signal level during the initial cycles of PCR, usually cycles 3 to
15, in which there is little change in fluorescent signal. The low-level signal of the baseline can be equated to the background or
the “noise” of the reaction
Baseline
The _____ of the real-time PCR reaction is the level of signal that reflects a statistically significant increase over
the calculated baseline signal
threshold
The ______ at which the amplification plot crosses the threshold (i.e., there is a significant detectable increase in
fluorescence).
CT can be a fractional number and allows calculation of the starting template amount
- Cycle Threshold
There are two types of DNA analysis in
qPCR
- ds DNA binding dye
chemistry - fluorophore-linked probes
_____ for both specific and nonspecific
detection of amplified products
ds DNA binding dye
chemistry
for specific PCR product detection
fluorophore-linked probes
commonly used; unsuitable for high-resolution melt curve analysis (HRM)
SYBR Green
less inhibitory to PCR; suitable forqPCR using a fast cycling protocol
EvaGreen
This analysis consists of applying heat to the sample (from 50°C to 95°C) and monitoring the fluorescence emission
during the process
Melting Curve Analysis
- are small fluorescent molecules that are attached to oligonucleotides in
order to function as probes
Fluorophores
There are two types of fluorophores:
donor/reporter and acceptor/quencher
Types of Fluorophores Used in qPCR
- Oligonucleotides that combine a primer and probe in a single molecule.
- They can be classified into three groups: Harpins, Cyclicons and Angler® primerprobes.
- Fluorescence emitted from primer-probes is detected and measured during the
denaturation or extension phase of the qPCR, depending on the type of primer-probe
used
Primer probes
detected and measured during the denaturation or extension phase of the qPCR, depending on the type of primer-probe
used
Fluorescence emitted from primer-probes
include Scorpions, Amplifluor, and Light-Upon-eXtension
(LUX)
Hairpin primer-probes
Oligonucleotides with an attached-donor and/or - acceptor fluorophore. There are two
types: hydrolysis and hybridization probes
Probes
Their mechanism of action relies on the 5′–3′ exonuclease activity of Taq polymerase, which degrades the bound probe during amplification.
Hydrolysis probes
