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RT PCR

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RT PCR

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hulwhulm
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RT-PCR

Uses RNA as starting material for in vitro nucleic acid amplification.


Reverse transcriptase (discovered in the early 1970s)

Reverse transcriptase is an RNA-dependent DNA polymerase, catalyzing DNA


synthesis using RNA as the template.
The end product - complementary DNA (cDNA).

Starting RNA is subsequently degraded, dsDNA is produced, and PCR amplification


proceeds in the usual manner. RNA extraction kits for both manual and automated
RNA purification exist and, when combined with RT-PCR, make RNA analysis in the
clinical laboratory virtually as rapid and equally sensitive as PCR-based DNA
amplification. RT-PCR is commonly used in the diagnosis and quantification of RNA
virus infections (e.g., human immunodeficiency virus and hepatitis C virus) and the
analysis of mRNA transcripts such as those produced by translocations commonly
associated with non-Hodgkin's lymphomas, leukemias, and sarcomas. Gene
expression profiling is likely to have a major impact on molecular diagnostics in the
coming years and will depend on RNA analysis using RT-PCR and possibly
high-density arrays.
RT-PCR (reverse transcription-polymerase chain reaction) is the most sensitive
technique for mRNA detection and quantitation currently available. Compared
to the two other commonly used techniques for quantifying mRNA levels,
Northern blot analysis and RNase protection assay, RT-PCR can be used to
quantify mRNA levels from much smaller samples. In fact, this technique is
sensitive enough to enable quantitation of RNA from a single cell
Real-time PCR: Principles and Applications

Real-time PCR also called quantitative PCR (qPCR), is a variant of standard polymerase
chain reaction in which amplification and simultaneous quantitation of a target DNA is
done in the same PCR machine, using commercially available fluorescence-detecting
thermocyclers. Fluorescent dyes specifically label DNA of interest, and the amount of
fluorescence generated is proportional to the quantity of DNA present.
Although various models of real-time PCR are available, all share common features: a
standard thermocycler platform coupled with an excitation source (usually a laser or
tungsten lamp), a camera for fluorescence detection, and computer and software for
data processing.
Depending on the available excitation source and detection filters, a variety of
fluorescent dyes may be used in qPCR. The two most commonly used real-time PCR
methods are SYBR green (a dye that binds to double-stranded DNA but not to
single-stranded DNA, and, when so bound, fluoresces) and TaqMan probes,
respectively.
Principle
Real-time PCR is accomplished in the same manner as conventional PCR-based
assays (denaturation of double-stranded DNA followed by primer annealing and
extension). However, it is the detection process that discriminates real-time PCR
from traditional PCR assays. In real-time PCR assays, the accumulation of amplicon
is monitored as it is generated using the labeling of primers with dyes capable of
fluorescence. These labels produce a change in fluorescent signal measured by the
instrument following their direct interaction with or hybridization to the amplicon.
This signal is related to the amount of amplified product present during each cycle
and increases as the number of specific amplicons increases.
Currently, a range of fluorescent chemistries are used for amplicon detection; the
more commonly used chemistries can be divided into two categories: (1) those that
involve the nonspecific binding of a fluorescent dye (e.g., SYBER Green I) to
double-stranded DNA and (2) fluorescent probes that bind specifically to the target
of interest.
Detection Methods
A number of real-time PCR methods have been described, but two have emerged
as the most popular.

PCR using SYBR


SYBR green is a dye that binds to double-stranded DNA but not to single-stranded
DNA, and, when so bound, fluoresces. During PCR cycle, as more and more
double-stranded product is generated to which SYBR green dye attach and
fluoresces, an increasing amount of fluorescent signal is generated. The amount of
fluorescence in the reaction at any particular time is directly related to the number
of double-stranded DNA molecules in the reaction.
However, the downside of SYBR green is that it will bind and fluoresce all
double-stranded products in the reaction, whether they are specific products,
nonspecific products, primer dimers, or other amplification artefacts.

TaqMan PCR (5’ nuclease assay)


TaqMan PCR uses dye-labelled nucleic-acid probe complementary to an internal
segment of the target DNA. This dye-labelled probe anneals to one of the template
strands close to and downstream from one of the two PCR primers. The probe is
labeled with two fluorescent moieties, the reporter (fluorophore) is attached to
the probe’s 5’ end and the quencher is attached to its 3’ end.
When the reporter and the quencher are connected, the quencher reduces the
fluorescent signal of the reporter dye as it absorbs the energy via a fluorescence
resonance energy transfer (FRET). However, during PCR, Taq polymerase, extending
the primer on the probe’s target strand, displaces and degrades the annealed
probe through the action of its 5’ to 3’ exonuclease function. The fluorophore is
thereby released from its molecular attachment to the quencher and fluoresces.
As more PCR products are generated, the more dye-labeled probe will find target
regions to join which eventually leads to release of the reporter molecule during the
subsequent amplification process. The release of reporter dyes is reflected as
increased intensity of the fluorescent signal which is proportional to the amount of
amplicon synthesized.
Whether using SYBR green or TaqMan probes, the relationship between signal
intensity and the amount of template in a real-time PCR reaction provides a reliable
means both to quantitate nucleic acids and to assay for the presence or absence of
specific gene sequences.
Applications
Real-time PCR enables calculation of the starting template concentration and is a
frequently used analytical tool in evaluating DNA copy number, viral load, SNP
detection, and allelic discrimination. When preceded by reverse-transcription PCR,
qPCR is a powerful tool to measure mRNA expression and is the gold standard
for microarray gene expression data confirmation.

Advantages
Significant advantages of real-time PCR include
its ability to measure DNA concentrations over a large range,
its high sensitivity,
its ability to process multiple samples simultaneously and providing immediate
information.
A disadvantage is the machines are more expensive than traditional PCR machines.
RT-PCR: Definition, Principle, Enzymes, Types, Steps, Uses

Polymerase chain reaction (PCR) is a temperature-dependent nucleic acid


amplification technique used to amplify the DNA or RNA in vitro enzymatically.

Developed by Kary Mullis and his associates at mid – 1980s, it is a very powerful and
most important tool in modern biology – molecular biology and genetics. It combines
the principle of nucleic acid hybridization with the principle of nucleic acid
replication. Using this non-culture-based nucleic acid amplification technique, we
can produce billions of copies of a single segment of DNA or RNA in a very short
time.

Since its development, several modifications have been made, and now there are
different types of PCR techniques available for different purposes. Reverse
transcriptase PCR and Quantitative PCR (qPCR) are the most commonly used PCR
types.
Reverse transcriptase polymerase chain reaction, RT-PCR, is a type of PCR
technique that enzymatically amplifies the RNA in vitro.

It is the only type of PCR that can amplify the RNA. It uses a reverse transcriptase
enzyme in addition to the other basic components of the PCR.

First, the sample RNA is converted to complementary DNA (cDNA) in reverse


transcription, catalyzed by the reverse transcriptase enzyme. These cDNA
molecules are then used as a template for amplification in the PCR process.

RT-PCR is used to analyze the mRNA or micro RNA and study gene expression.
Objectives of RT-PCR
To amplify the specific segment of RNA, resulting in billions of copies of a single RNA
segment.
To diagnose certain infections, genes, and study gene expression.

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