RT-PCR can be performed as either a two-step or an one-step
procedure. Each has certain advantages:
1. Two-step procedures
A. Two tube, two-step procedure.
In the first tube, first-strand cDNA synthesis is performed
under optimal conditions, using either random hexamers, oligo(dT) primers
(generating a cDNA pool), or sequence-specific primers. An aliquot of
the RT reaction is then transferred to another tube (containing thermostable
DNA polymerase, DNA polymerase buffer, and PCR primers) for PCR.
Advantages of this approach: This method is useful for
experiments where multiple transcripts have to be analyzed from the same
RT reaction or for specific applications such as Differential Display
Reverse Transcription (DDRT) or Rapid Amplification of cDNA Ends (RACE).
Also, since the RT reaction is performed under optimal conditions, this
approach produces the longest RT-PCR products (up to 14 kb in length,
if the appropriate enzymes are used).
B. One tube, two-step procedure.
In the first step, reverse transcriptase produces first-strand cDNA in
the presence of Mg2+
ions, high concentrations of dNTPs, and
either specific or non-specific [oligo(dT)] primers
volume, 20 l). Following the RT reaction, an optimized PCR buffer (without
ions), a thermostable DNA polymerase, and specific primers
added to the tube and PCR is performed. This approach may
be useful when
template amounts are limited, since the entire
RT reaction is used in the
Advantages of Two-step RT-PCR Procedure.
A two-step procedure has the following advantages:
Optimizes reaction conditions. The two-step format
allows both reverse transcription and PCR to be performed under optimal
conditions to ensure efficient and accurate amplification.
Provides flexibility. Two-step procedures allow the
product of a single cDNA synthesis reaction to be used for analysis
of multiple transcripts. This flexibility is valuable for such specialized
applications as rapid amplification of cDNA ends (RACE) and Differential
Display Reverse Transcription (DDRT).
2. One tube, one-step procedure (coupled RT-PCR)
Amplifies long sequences. With the right combination
of reverse transcriptase and thermostable DNA polymerase, two-step
RT-PCR can amplify RNA sequences up to 14 kb long.
Both cDNA synthesis and PCR amplification are performed with the same
buffer and site-specific primers, eliminating the need to open the reaction
tube between the RT and PCR steps. In addition to the higher sensitivity
of this approach (as in the one tube, two-step reaction above), the one-step
approach minimizes the chance of contamination, since the entire reaction
is performed with minimal pipetting steps and without opening the tube.
In addition, this approach permits direct analysis of a specific transcript,
since the primers used in both steps are sequence-specific. Finally, the
thermoactive reverse transcriptase used in this procedure allow a high RT
reaction temperature (5072C), which reduces false priming and increases
the specificity of the reaction by eliminating secondary mRNA structure.
Advantages of One-step RT-PCR Procedure.
A one-step procedure has the following advantages:
Minimizes time required. The one-step reaction has
fewer pipetting steps than the two-step reaction, significantly reducing
the time needed to perform RT-PCR and eliminating pipetting errors.
Reduces the risk of contamination. The entire one-step
reaction takes place in a single tube, with no transfers required
and no need to open the reaction tube (steps where contamination of
the PCR sample can take place).
- Improves the sensitivity and specificity of cDNA synthesis. Two characteristics
of the one-step reaction provide increased yield and efficiency: (1)
the cDNA reaction is performed at a high temperature (to eliminate problems
with RNA secondary structure), and (2) the entire cDNA sample is used
as template for PCR.
Source:Page: All 1 2 3 Related biology technology :1
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