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Controls for PCR WHY DO WE NEED CONTROLS FOR PCR?? 

• 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 THANK YOU