Biology Reference
In-Depth Information
Table 8.1: Example of a Standard Allele-Specific PCR Reaction Protocol for Amplifying
Drosophila
DNA.
1.
Set up a 100-
μ
l reaction in a 0.5-ml microfuge tube, mix, and overlay with 75
μ
l of mineral oil:
Template DNA (10
5
-10
6
target molecules)
20 pmol each primer (each primer 18-30 nucleotides long)
100 mM Tris-HCl (pH 8.3 at 20°C)
10 mM MgCl
2
0.05% Tween 20
50
μ
M each dNTP
2 units of
Taq
DNA polymerase
2.
Perform 25-35 cycles of PCR using the following temperature profile:
Denaturation
96 °C, 15 seconds
Primer annealing
55 °C, 30 seconds
Primer extension
72 °C, 1.5 minutes
3.
Cycling should conclude with a final extension at 72 °C for 5 minutes. Stop reactions by chilling to 4 °C
and/or adding ethylene dinitrilotetra-acetic acid (EDTA), a chelating agent to 10 mM.
Figure 8.2
Example of a typical PCR protocol. Step 1 involves denaturating the double-stranded DNA
template at 94 °C. Step 2 involves annealing the primer to the single-stranded target DNA by base
pairing. Step 3 involves synthesis of new DNA from the 3
′
end of the primer (
=
primer extension) by
DNA polymerase using dNTPs in a sequence determined by the template DNA. Steps 1-3 are a cycle,
and approximately 25 cycles will yield an increase in DNA content by a factor of
≈
1 million (2
25
).
each cycle (
Saiki et al. 1988, Eckert and Kunkel 1991, Taylor 1991, Goodman
1995
).
Taq
was isolated from the bacterium
Thermus aquaticus
that was col-
lected from a hot spring in Yellowstone National Park. Because
Taq
can with-
stand repeated exposures to temperatures up to 94 °C, its use greatly increased
the ease with which the PCR could be performed.
