• PCR amplification is a highly error and contamination
prone technique.
• PCR controls ensure the accuracy and reliability of any
PCR reaction.
What are Controls
• Controls are some pre-set parameters using which we
will interpret our PCR results.
PCR controls are used to
• Validate the performance of the test.
• Validate the results of the test.
• Evaluate reasons for false or negative results.
• Troubleshooting the problems.
• Give strength and scope to optimize other reactions.
TYPES OF CONTROLS
• Blank/No Template Control(NTC)
• Positive control
• Negative control
• Standard control
• Template control
• Reaction control
Blank/No Template Control(NTC)
• Contains all the reagents, but no DNA template
• To check for contamination/false positive results.
• Instead of template DNA, nuclease-free water is added to the
reaction which also validates whether the water is of high quality or
not.
• PCR enhancers can also be added, if they are used in the main
reaction to validate the purity of enhancers.
Negative control(NC)
• Contains all the reagents and DNA template , but no target sequence.
• Control for specificity (true negative).
• Negative control reactions can also be used to check for primer selfdimers (homo-dimers).
Positive control(PC)
• Contains all the reagents and DNA template with target sequence.
• Control for sensitivity (true positive), there are no PCR inhibitors
Internal control
• Internal control also known as internal positive control
or internal amplification control (IAC) is a reactionindependent control.
• The internal control is designed in a way that coamplifies the genomic region other than our target, using
the same annealing temperature, reaction conditions and
ingredients (except primers).
Internal control types
• Exogenous controls and Endogenous controls.
• The endogenous internal controls which are typically housekeeping
genes amplify along with the template showing whether the starting
template has a sufficient quantity or not.
• Endogenous controls are targets within the studied sample, other
than the region/regions of interest, such as constitutively expressed
genes serving basal cell functions, and are therefore dependent on
the type tissue being examined.
Internal control
• Exogenous controls are pipetted directly into the raw sample, or
added to the isolated nucleic acid before the PCR reaction and will
target a different species to the studied one.
• They are mainly used for absolute quantification (i.e. number of gene
copies per examined sample) and for the control of the relative
amplification from a set of specific primers.
• Another use for exogenous controls is the discrimination between
negative samples (where the control will still amplify) and PCR
inhibition (when no amplification will be observed).
• Synthetic noncompetitive exogenous control is generally used for
microbial detection.
REFERENCES
• https://geneticeducation.co.in/type-of-pcr-controls-negative-positiveand-internalcontrols
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