Introduction to the Polymerase Chain Reaction - Clinical Applications of PCR - page 4

5. Steps of the PCR
5.1. Thermal Denaturation
A common cause of failed PCR is inadequate denaturation of the DNA tar-
get. We typically use an initial denaturation temperature of 94°C for 8 min.
Modified Taq polymerase, e.g., Ampli Taq Gold ® , can be activated during this
initial denaturing process to achieve a Hot Start PCR. For subsequent cycles,
94°C for 1-2 min is usually adequate. As the targets of later PCR cycles are
mainly PCR products rather than genomic DNA, it has been suggested that the
denaturation temperature may be lowered after the first 10 cycles so as to avoid
excessive thermal denaturation of the Taq polymerase ( 6 ) . The half-life of Taq
DNA polymerase activity is more than 2 h at 92.5°C, 40 min at 95°C, and
5 min at 97.5°C.
5.2. Primer Annealing
The temperature and length of time required for primer annealing depends
on the base composition and the length and concentration of the primers. Using
primers of 18-30 bases long with approx 50% GC content and an annealing
step of 55°C for 1-2 min is a good start. In certain primer-template pairs, a
difference in the annealing temperature as small as 1-2°C will make the differ-
ence between specific and nonspecific amplification. If the annealing tempera-
ture is >60°C, it is possible to combine the annealing and extension step together
into a two-step PCR cycle.
5.3. Primer Extension
Primer extension is typically carried out at 72°C, which is close to the
optimum temperature of the Taq polymerase. An extension time of 1 min is
generally enough for products up to 2 kb in length. Longer extension times
(e.g., 3 min) may be helpful in the first few cycles for amplifying a low copy
number target, or at later cycles when product concentration exceeds enzyme
concentration.
6. Cycle Number
The number of cycles needed is dependent on the copy number of the target.
As a rule of thumb, amplifying 10 5 template molecules to a signal visible on an
ethidium bromide-stained agarose gel requires 25 cycles. Assuming that we
use 1 min each for denaturation, annealing, and extension, the whole process
can be completed in approx 2-3 h (with extra time allowed for the lag phase
taken by the heat block to reach a certain temperature). Similarly, 10 4 , 10 3 , and
10 2 target molecules will require 30, 35, and 40 cycles, respectively.