Biology Reference
In-Depth Information
inhibit the PCR. Too little template DNA can result in false negatives (
Rameckers
et al. 1997
). The number of template molecules and cycles needed to give a
good yield (
≈
10 ng of DNA) was estimated, making the assumption that the effi-
ciency of the PCR actually is approximately 70% (not 100%) and the product is
200 bp in length (
Rameckers et al. 1997
):
No. of template DNA molecules
Theoretical no. of cycles required
1
44
10
40
100
35
1,000
31
10,000
27
100,000
22
The efficiency of DNA amplification declines in the later cycles. This is called
the
amplification plateau
because the product stops being produced exponen-
tially and enters a linear or stationary phase (
Kainz 2000
). The plateau appears to
be due to the binding of DNA polymerase to its amplification products. In gen-
eral, it is better to set up multiple reactions if large amounts of DNA are needed.
8.3.13 Reducing the Evils of Contamination
It is crucial that laboratory techniques be meticulous to prevent contamination
of the laboratory, supplies, and equipment with target DNA. Contamination can
be an enormous problem because allele-specific PCR can generate copies of DNA
from very small amounts of template (theoretically from a single molecule).
Carryover of tiny quantities of PCR product can lead to
false positives
in subse-
quent reactions.
There is no simple and guaranteed method to prevent contamination. You
must be thoughtful and careful
at all times
, using a variety of approaches to
reduce
the possibilities of contamination. Most importantly, you must have ade-
quate controls to
detect
contamination.
Work surfaces can be decontaminated with 0.07 M sodium hypochlorite (10%
bleach), which degrades DNA. Commercial products containing RNase solution
eliminates RNA. UV irradiation of the workstation can be helpful, although
dried DNA is less susceptible to UV irradiation than hydrated DNA (
Roux 1995
).
Don't forget that UV irradiation of DNA polymerases, DNA template, and prim-
ers can damage them, reducing the efficiency of the PCR. UV light was reported
to inhibit PCR amplification efficiency, even when only the water was irradi-
ated, so routine decontamination with UV light should be used cautiously
(
Pao et al. 1993
).
