(RNA Template-Specific PCR, RS-PCR)
Transcript of (RNA Template-Specific PCR, RS-PCR)
Biotechnology-Theory Lec. 4 3rd Student-Medical Analysis 2021
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REVERSE TRANSCRIPTASE PCR (RT-PCR)
(RNA Template-Specific PCR, RS-PCR)
Sensitive method for the detection and analysis of rare mRNA transcripts or
other RNAs present in low abundance are an important aspect of most cell/
molecular biology studies. RNA cannot serve as a template for PCR, so it must
first be reverse transcribed into DNA known as the" complementary DNA or
cDNA." Powell et al. first described a combined technique, now commonly
known as RT-PCR in which reverse transcription (RT) is coupled with PCR
amplification of the resulting cDNA.
Reverse transcription polymerase chain reaction (RT-PCR): is one of
many variants of polymerase chain reaction (PCR). This technique is commonly
used in biotechnology qualitatively to detect gene expression through creation of
complementary DNA (cDNA) transcripts from RNA. It is an extraordinarily
sensitive method to detect as few as l-100 copies of a specific RNA. This is also
achieved by monitoring the amplification reaction using fluorescence, a technique
called real-time PCR or quantitative PCR (qPCR). Combined RT-PCR and qPCR
are routinely used for analysis of gene expression and quantification of viral RNA
in research and clinical settings.
Analysis of gene expression requires accurate determination of mRNA levels.
But PCR is based on amplification of DNA rather than RNA, so how can it be
used for mRNA analysis? The answer is that first, mRNA is converted into DNA
using the well-known process of reverse transcription, which is used by RNA
viruses to convert their genomic RNA into a DNA within the host cell; and
second, PCR amplification is performed on the resulting complementary DNA
(cDNA).
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Amplification of mRNA molecules to study gene expression can be achieved
by a method that combines two sequential enzymatic steps: the synthesis of DNA
from the RNA template using dNTPs by an RNA-dependent DNA polymerase
[reverse transcriptase] (RTase enzyme) derived by AMV (Avian Myoblastosis
Virus) reverse transcriptase or Thermus thermophilus (Tth) DNA polymerase with
using Oligo dT primer or random hexamer primer and followed by PCR using of
a heat stable DNA polymerase (Taq polymerase). The extremely high sensitivity
of RT-PCR enables us to detect rare mRNAs, mRNAs in small numbers of cells
or in small amounts of tissue as well as mRNAs expressed in mixed-cell
populations.
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This enzyme is a RNA dependent DNA polymerase able to perform the
following reactions:
ο Synthesizes a DNA strand on an RNA template.
ο Removes the RNA strand from the DNA: RNA duplex (RNase H activity).
ο Synthesizes a second DNA strand on the DNA template.
This enzyme is used in the replication cycle of Retroviruses, a group of viruses
that converts their RNA genomes into DNA that will then integrate in the genomes
of host cells. The enzyme preparations that are commercially available are
therefore typically of retroviral origin, the most common of which are avian
myeloblastosis virus (AMV) reverse transcriptase and Moloney murine leukemia
virus (M-MuLV) reverse transcriptase. Tth polymerase, an enzyme isolated from
the bacterium Thermus thermophilus, is a heats table DNA polymerase.
The AMV and m-MuLV viral RTases are highly processive and are able to
synthesize cDNAs of up to 10 kb. Tth DNA Polymerase is able to synthesize
cDNA in the range of 1.0 - 2.0 kb, which is sufficient since fragments of <1 kb
are usually used for PCRs. The unique advantage of the Tth DNA Polymerase is
its ability to perform both reverse transcription and PCR amplification in a one-
step reaction.
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RT PCR is a two-step process:
1) In the first step of RT-PCR, called the "first strand reaction,"
complementary DNA is made from a messenger RNA template using dNTPs
and an RNA-dependent DNA polymerase (reverse transcriptase) through the
process of reverse transcription. RT-PCR exploits a characteristic of mature
mRNAs known as the 3' polyadenylated region, commonly called the poly (A)
tail, as a common binding site for poly (T) DNA primers. In the case of
bacterial mRNA, which lack a poly (A) tail sequence-specific primers can be
generated to amplify the target mRNA sequence. These primers will anneal to
the 3' end of every mRNA in the solution, allowing 5'-3' synthesis of
complementary DNA by the reverse transcriptase enzyme. Complementary
DNA can also be prepared from mRNA by using gene specific primer or
random hexamer primers.
2) After the reverse transcriptase reaction is complete, and complementary DNA
has been generated from the original single stranded, standard polymerase
chain reaction, termed the "second strand reaction," is initiated.
After approximately 30 cycles, millions of copies of the sequence of
interest are generated. The original RNA template is degraded by RNase H,
leaving pure cDNA (Plus spare primers).
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I. Two step RT-PCR. The synthesis of cDNA is performed with reverse
transcriptase from AMV, moleney murine leukemia virus (M-MuLV), or
Thermus thermophilus (Tth) DNA polymerase in the first step, followed by
PCR with an appropriate thermostable DNA polymerase. The two-step
reaction requires that the reaction tube is opened after cDNA synthesis and
reagents are added for the PCR part of the procedure. This is inconvenient and
increases the risk of contamination.
II. One-step RT -PCR. The cDNA synthesis and the PCR are performed together
in a single tube. Two techniques are in use for running one-step RT-PCRs. The
first technique uses Tth DNA Polymerase for carrying reverse transcription and
PCR reactions. The second uses AMV reverse transcriptase and Taq DNA
Polymerase.
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There are three types of primers that may be used for reverse transcription:
1. Oligo (dT) [deoxy-thymine nucleotides] 12-18 primer. This primer binds to the
endogenous poly (A) +tail at the 3’-end of mammalian mRNA. A reaction with this
primer frequently produces a full-length cDNA product.
2. Random Hexanucleotide Primers. These primers can bind to mRNA templates at
any complementary site and will give partial length (short) cDNAs. These primers
may be better for overcoming the difficulties caused by template secondary structure.
The random primers may also transcribe more 5' - regions of the RNA.
3. Specific Oligonucleotide Primers. These primers can be used for selectively
priming the RNA of interest. This approach has been used very successfully in
diagnostic assays, as well as in basic research.
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RT-PCR has found significant application in the field of disease diagnostics
and some of these applications in the field of infectious diseases.
ѽ Many clinically important viruses have genomes composed of RNA, RT-PCR
is useful for detecting such viruses. RT-PCR has also been used for the
detection of the viral causes of meningitis and meningoencephalitis, such as
enteroviruses and the West Nile virus. RT-PCR is being used for the detection
of the following viruses: Dengue virus, Hantavirus, Human metapneumovirus,
Severe acute respiratory syndrome (SARS) and Coronavirus (COVID-19).
ѽ Quantitative RT-PCR assays are commonly used for the detection of HIV and
HCV viral load (amount of these viruses present in the blood of a patient)
testing.
ѽ RT-PCR may also be used to detect other microorganisms (bacteria, parasites,
and fungi) by targeting their rRNA. This approach is better than detection of
DNA, as the presence of RNA is more likely associated with the presence of
viable organisms.
ѽ Detection of mRNA using RT-PCR helps to study the gene expression of both
microorganisms and human host cells.