Biomedical Engineering Reference
In-Depth Information
Cycle number
Number of double-stranded
target molecules
fere with the PCR, and ways of eliminating them
have been reviewed by Bickley and Hopkins (1999).
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
0
0
2
4
8
16
32
64
128
256
512
1024
2048
4096
8192
16,384
32,768
65,536
131,072
262,144
524,288
1,048,576
2,097,152
4,194,304
8,388,608
16,777,216
33,554,432
67,108,864
134,217,728
268,435,456
RT-PCR
The thermostable polymerase used in the basic PCR
requires a DNA template and hence is limited to the
amplification of DNA samples. There are numerous
instances in which the amplification of RNA would
be preferred. For example, in analyses involving the
diffierential expression of genes in tissues during
development or the cloning of DNA derived from an
mRNA (complementary DNA or
cDNA
), particularly
a rare mRNA. In order to apply PCR methodology
to the study of RNA, the RNA sample must first be
reverse-transcribed to cDNA to provide the necessary
DNA template for the thermostable polymerase. This
process is called reverse transcription (RT), hence
the name RT-PCR.
Avian myeloblastosis virus (AMV) or Moloney
murine leukaemia virus (MuLV) reverse transcrip-
tases are generally used to produce a DNA copy of
the RNA template. Various strategies can be adopted
for first-strand cDNA synthesis (Fig. 2.9).
Fig. 2.8
Theoretical PCR amplification of a target fragment
with increasing number of cycles.
Long accurate PCR (LA-PCR)
Amplification of long DNA fragments is desirable for
numerous applications of gene manipulation. The
basic PCR works well when small fragments are
amplified. The efficiency of amplification and there-
fore the yield of amplified fragments decrease signi-
ficantly as the size of the amplicon increases over 5 kb.
This decrease in yield of longer amplified fragments
is attributable to partial synthesis across the desired
sequence, which is not a suitable substrate for the
subsequent cycles. This is demonstrated by the pres-
ence of smeared, as opposed to discrete, bands on a gel.
Barnes (1994) and Cheng
et al
. (1994) examined
the factors affecting the thermostable polymeriza-
tion across larger regions of DNA and identified key
variables affecting the yield of longer PCR frag-
ments. Most significant of these was the absence of
a 3
Recent developments have sought to minimize
amplification times. Such systems have used small
reaction volumes in glass capillaries to give large
surface area-to-volume ratios. This results in almost
instantaneous temperature equilibration and minimal
annealing and denaturation times. This, accompan-
ied by temperature ramp rates of 10 -20°C/s, made
possible by the use of turbulent forced hot-air sys-
tems to heat the sample, results in an amplification
reaction completed in tens of minutes.
While the PCR is simple in concept, practically
there are a large number of variables which can
influence the outcome of the reaction. This is espe-
cially important when the method is being used with
rare samples of starting material or if the end result
has diagnostic or forensic implications. For a detailed
analysis of the factors affecting the PCR, the reader
should consult McDowell (1999). There are many
substances present in natural samples (e.g. blood,
faeces, environmental materials) which can inter-
exonuclease (proofreading) activity in
Taq
polymerase. Presumably, when the
Taq
polymerase
misincorporates a dNTP, subsequent extension of
the strand either proceeds very slowly or stops
completely. To overcome this problem, a second
′
-5
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