Biomedical Engineering Reference
In-Depth Information
Box 2.3 The polymerase chain reaction achieves enormous amplifications,
of specific target sequence, very simply
The reaction is assembled in a single tube, and then
placed in a thermal cycler (a programmable
heating/cooling block), as described below.
A typical PCR for amplifying a human genomic
DNA sequence has the following composition. The
reaction volume is 100
m
l.
Input genomic DNA, 0.1-1
m
g
Primer 1, 20 pmol
Primer 2, 20 pmol
20 mmol/l Tris-HCl, pH 8.3 (at 20°C)
1.5 mmol/l magnesium chloride
25 mmol/l potassium chloride
50 mmol/l each deoxynucleoside triphosphate
(dATP, dCTP, dGTP, dTTP)
2 units
Taq
DNA polymerase
A layer of mineral oil is placed over the reaction
mix to prevent evaporation.
The reaction is cycled 25-35 times, with the
following temperature programme:
Denaturation 94°C, 0.5 min
Primer annealing55°C,1.5 min
Extension 72°C, 1 min
Typically, the reaction takes some 2-3 h overall.
Notes:
• The optimal temperature for the annealing step
will depend upon the primers used.
• The pH of the Tris-HCl buffer decreases markedly
with increasing temperature. The actual pH varies
between about 6.8 and 7.8 during the thermal cycle.
• The time taken for each cycle is considerably
longer than 3 min (0.5
1 min), depending
upon the rates of heating and cooling between steps,
but can be reduced considerably by using turbo
systems (p. 21).
• The standard PCR does not efficiently amplify
sequences much longer than about 3 kb.
1.5
+
+
Random primer
5'
AAAAAAA 3'
mRNA
1st strand cDNA
3'
random primer
Oligo (dT) primer
5'
AAAAAAA
3'
mRNA
1st strand cDNA
3'
TTTTTTT 5'
Sequence-specific primer
Fig. 2.9
Three strategies for synthesis of
first-strand cDNA. (a) Random primer;
(b) oligo (dT) primer; (c) sequence-specific
primer.
5'
AAAAAAA
3'
mRNA
1st strand cDNA
3'
primer
thermostable polymerase with proofreading capab-
ility is added. Thermostable DNA polymerases with
proofreading capabilities are listed in Table 2.1.
• Primers should be 17 to 30 nucleotides in length.
• A GC content of about 50% is ideal. For pri-
mers with a low GC content, it is desirable to
choose a long primer so as to avoid a low melting
temperature.
• Sequences with long runs (i.e. more than three or
four) of a single nucleotide should be avoided.
• Primers with significant secondary structure are
undesirable.
Key factors affecting the PCR
The specificity of the PCR depends crucially upon the
primers. The following factors are important in
choosing effective primers.
