Inhibition and Facilitation

of Nucleic Acid Amplification

IAN G. WILSON
 Inhibition and Facilitation
of Nucleic Acid Amplification
Polymerase chain
reaction (PCR) was the
first nucleic acid
amplification method Nucleic Acid Amplification Test: technique
developed and until now has used to detect a particular nucleic acid, virus,
been the method of choice or bacteria, which acts as a pathogen
 Steps for Polymerase Chain Reaction
1. Denaturation (96°C): Heat the reaction to separate the DNA
strands
2. Annealing (55-65°C): Cool the reaction so the primers can bind to
their complementary sequences on the single strand of DNA
3. Extension (72°C): Raise the reaction temperatures so Taq
polymerase extends the primers, synthesising new strands of DNA
Inhibition and Facilitation of Nucleic Acid Amplification
False positives: a test result which incorrectly indicates that a particular
condition or attribute is present.
Factors that affect detection levels:
•Dilution of samples The minimum number of cells containing a single-
•Intracellular bacteria may copy target gene theoretically detectable in a
10-ml sample aliquot added to a 90-ml reaction
be more sensitive than mix is one.
expected because of
This figure corresponds to 100 CFU ml-1
multiple bacterial cells in
each infected blood cell
•Unclear about whether the
number of cells detectable is
per millilitre or per reaction
volume
 Reaction Conditions for PCR
•Excellent thermal consistency is important for sensitive and specific
testing
•Dimethyl sulfoxide (DMSO) has been shown to improve reaction yield
during RT-PCR
• Concentration of Mg2+ ions will affect the success and specificity of

amplification.
 Contamination and Other Factors
•Inhibition can arise from other endogenous sources, including reaction
components.
•Commercial preparations of Taq polymerase, including a low-level-DNA
product, have been shown to be contaminated with eubacterial DNA
that originates from neither E.coli nor Thermus aquaticus.
•Cellulose and nitrocellulose filters were found to inhibit PCR
•Exogenous contamination also occurs with the same mechanisms.
Exogenous & Endogenous: developing for
out/inside factors