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REVERSE TRANSCRIPTION-POLYMERASE C H A I N
REACTION: A N OVERVIEW OF THE T E C H N I Q U E
A N D ITS APPLICATIONS
Abstract
Keywords
Introduction
PCR allows the sensitive, selective detection and amplification of a target DNA
fragment (43). It involves the initial denaturation of the DNA, followed by the
13
14 N.W. OHAN and J. J. HEIKKILA
annealing of target-specific oligonucleotide primers to each strand of DNA, and
finally the extension of the primers to produce a copy of the selected region.
By r e p e a t i n g this cycle of denaturation, primer annealing and primer
extension, there is an exponential amplification of the target. In theory, after
20 cycles, there could be 220 , or over one million copies of the target produced.
Rapid advances are being made in PCR technology (2,15, 53, 34). An extension
of the technique involves the addition of a reverse transcription step prior to
PCR, allowing one to apply the power of PCR to RNA (Fig. 1) (40,24,27,48). This
procedure has been variously referred to as RT-PCR, RNA phenotyping,
RNA/PCR and message amplification phenotyping (MAPPing). This overview
presents the various components comprising RT-PCR, including methods of
RNA isolation, reverse transcription, PCR reagents, primer design and dealing
with c a r r y - o v e r contamination, among others. Finally, some of the recent
applications of RT-PCR are examined. These include quantitative analysis of
mRNA levels via competitive RT-PCR and a relatively new variation of RT-PCR
which allows the study of differential eukaryotic mRNA transcription.
RNA Isolation
0
Guanidine
p-
A M V - R T OR MmLV-RT
Oligo (dT)/Random
p- c D N A for MR
Yhioeyanate/CsCl
Hexamers/SpeciflcPrimers
Method
I~ O L ~ S E
CHAINREACTION I
I
CYCLE 1 r''~
i
I
CYCLE2 CYCLE 3
DENATURATION 3' TTT5': 3' TTT5' 3T TTT5'
~NA 5'-- TTT3,
15'-- rrTylI
'Long'Product 5'-- TTT 3'
3,__..-........~,5,
,L
5 ~ T T 3, 5 ' ~ T T 3,
3,1--......,,~ 5,
3 ' ~ T T 5'
PRIMER 3' + - - I t : : t : : ~ T T 5' 3 ' ~ T T 5' 5' -~T'r'rrr=r'ffTT 3,
EXTENSION 5'~;x~'T, TT3'
!
i 5' ~ ] ~ : G ~ I - T T T 3,
*
0 ......... ,
3 ' ~ 5 '
Repeat
,+++"+
+,r~or ,++==+l
3,~+ j
o °o= PCR Products:
- verify l e n g t h
more
I----I
- Southems
- Restriction digest
- Sequ~ce
Reverse Transcription
Once the cDNA has been synthesized, RT-PCR is subject to the same
parameters as regular 'DNA' PCR. However, there are some RT-PCR-specific
factors which must be addressed with respect to primer design and
contamination problems. The PCR and RT-PCR variables are briefly described
below. These include the concentration of reagents, selection of a DNA
polymerase, the thermal profile, product analysis, primer design and the
prevention of contamination. Once organized, RT-PCR can be completed in a
single day - from reverse transcription to visualization of the PCR products
following electrophoresis.
The concentrations of the PCR reagents depend on the DNA polymerase, PCR
primers and target length, and thus must be optimized separately for each
case. General PCR protocols (24) provide a good starting point for optimization
of the reaction.
The original DNA polymerase used in PCR to catalyze the extension of the
primers was the Klenow fragment of E. coli DNA polymerase I (14). The
problem with the Klenow polymerase was that it was denatured at the
beginning of each cycle and thus had to be continually replaced. The discovery
of thermostable DNA polymerases, able to withstand normal denaturing
temperatures, revolutionized the polymerase chain reaction by facilitating its
automation (43). The most commonly used thermostable polymerase is the
Taq DNA polymerase. Other polymerases are also available; for example, there
is the Vent DNA polymerase, which has a 3'-->5' proof-reading exonuclease
activity not possessed by Taq (13), making it more useful in sequencing
reactions because of its higher fidelity. As mentioned before the T t h
polymerase is also available and is capable of both reverse transcription and
PCR (36).
PCR products can be visualized after electrophoresis using either 1-2% agarose
or 6-10% polyacrylamide gels and ethidium bromide staining of the DNA
fragments. This procedure will verify that the synthesized DNA fragment is
the expected size. However, to unquestionably verify that the fragment is
indeed the targeted one, it must be subjected to at least one of the following
tests: (A) restriction digestion of the fragment using a known unique enzyme
site, (B) Southern hybridization analysis of the PCR fragments using a specific
probe or, (C) sequencing of the fragment.
Generally, the pair of primers are both between 20 and 30 bp in length with
similar A/T and G/C composition. Their calculated annealing temperatures
should be as close to 72°C as possible for optimal stringency. The higher the
stringency, the less likely mis-priming will occur, thereby minimizing non-
specific a m p l i f i c a t i o n . Annealing temperatures can be c a l c u l a t e d using
REVERSETRANSCRIPTION-PCR 19
empirically derived formulas (49,1,3,17,33,42,54). The formula employed by
the commercial "OLIGO" program is probably the most sophisticated (41,42).
The free-ware program "Oligonucleotide Synthesizing Program" (OSP) has also
met with success (22). After primers have been designed, formamide (44) and
d i m e t h y l s u l f o x i d e c o n c e n t r a t i o n s (16), which r e d u c e hydrogen bond
stabilities, can also be used to adjust annealing stringencies.
RT-PCR Applications
The high sensitivity of RT-PCR, coupled with the extremely low RNA quantities
required, has made it the most powerful method available for the detection of
rare transcripts. Some of its recent applications are listed in Table 1. A
possible problem with RT-PCR is that it is t o o sensitive. Chelly et aL (6) have
reported the detection of the expression of tissue-specific genes in non-
specific cells a phenomenon they term 'illegitimate transcription'. The
potential e x i s t e n c e of this basal expression suggests that a method of
quantitation using RT-PCR would be useful to distinguish different levels of
REVERSE TRANSCRIPTION-PCR m
Quantitative RT-PCR
The PCR reaction proceeds in an exponential manner until reagent limits are
met (12). If one can determine the final quantity of products (X), and one
knows the amplification efficiency (E, max. value = 1), and the number of
cycles (n), the initial amount of starting template (I) can be calculated using
the following equation (19):
X = I(I +E) n
The final yield, (X), can be determined from densitometric scans of ethidium
bromide stained gels (19), or the radioactive labelling of a primer or dNTP in
the PCR reaction mix which can then be quantitated via scintillation counting
of excised bands or d e n s i t o m e t r i c s c a n n i n g of its a u t o r a d i o g r a m
(35,7,12,19,40). Finally, the yield of PCR product can also be obtained by
Southern blot analysis, followed by the densitometric scanning of the resultant
autoradiogram (7,5).
Concluding Remarks
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