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Nested RT-PCR in a Single Closed Tube 151
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Nested RT-PCR in a Single Closed Tube
Antonio Olmos, Olga Esteban, Edson Bertolini, and Mariano Cambra
1. Introduction
There are several methods now in widespread use for detecting and characterizing
specific RNA targets. These methods include in situ hybridization, Northern blotting,
dot or slot blot, RNase protection assay, and reverse transcription coupled to polymerase
chain reaction (RT-PCR). However, when the amount of RNA target is limited or
restricted in its cellular or tissue distribution, the extreme sensitivity of the PCR
allows the detection of minute quantities of RNA when coupled to an initial step that
converts single-strand RNA to cDNA (1–4). Nevertheless, when RT-PCR is applied
for diagnostic purposes, the sensitivity usually afforded by this technique in routine
detection tests is similar, or only slightly higher, to conventional enzyme-linked
immunosorbent assay or hybridization techniques. This is frequently observed when
dealing with poor-quality samples containing inhibitors of RT-PCR. The presence of different
components of plant or animal origin, as well as specific RT-PCR conditions, may
inhibit the reverse transcription and amplification by a number of mechanisms (5).
Approaches have been developed to overcome these problems, including a previous
immunocapture phase (6–8), the immobilization of RNA targets on plastic surfaces (9) or
on paper membranes by a direct printing or squashing of the sample (10–12), preparation
of crude extracts in simple buffers and subsequent dilution in sterile water, and other
different protocols to prepare RNA targets free from interfering substances. Moreover,
there are interesting alternatives to crude extracts or total nucleic acid preparations based
on the use of commercially available resin- (13) or silica- (14) based kits.
Sensitivity and specificity problems associated with RT-PCR may be overcome by
using nested RT-PCR. The process is based on two consecutive rounds of amplification
(15,16), the first round being an RT-PCR and the second a conventional PCR. The
RT-PCR is performed using a pair of external primers. The 3′ end external primer (Pe1
in Fig. 1) is used for RT reaction and the 3′ and 5′ end (Pe2 in Fig. 1) primers are
used to perform the first PCR. The resulting amplification product is transferred to
another Eppendorf tube containing a second pair of nested primers (nested PCR)
that are internal to the initial pair. Alternatively, one of the external primers and a
single nested primer (heminested PCR) can also be used. The larger amplification
fragment produced during the first reaction is used as a target for the second (nested or
heminested) PCR. The concept is illustrated in Fig. 1.
From: Methods in Molecular Biology, Vol. 226: PCR Protocols, Second Edition
Edited by: J. M. S. Bartlett and D. Stirling © Humana Press Inc., Totowa, NJ
151