Type of PCR Controls- Negative, Positive and Internal Controls

Positive, Negative and Internal controls are common PCR control types employed as a reference to evaluate results. 


If you know PCR well, you probably understand why we use the controls! PCR controls have a vital role in evaluating results and finding errors during the run. To understand the PCR effectively one has to know why we use PCR controls and what’s its importance. 

PCR is a technique that needs ingredients like dNTPs, Taq DNA polymerase, PCR reaction buffer, template DNA and nuclease-free water. It’s an amplification method that copies a target DNA for various applications. 

Besides having many advantages, the technique has high sensitivity and so needs constant attention. Notedly, the assay varies among samples and requirements. In order to achieve success in every assay, we need to do everything precisely. 

And that’s why it needs optimization at every level. We are exploring PCR and PCR optimization in our new series, so far we have discussed the optimization of PCR cycles and annealing temperature. 

In the present article, I will explain what PCR controls are, their importance, and the types that we use in our PCR assays. I hope the present article will add more value to your PCR knowledge. 

Stay tuned, 

What is a PCR control?

Positive, Negative or internal control of PCR is used in the reaction to validate the results and let us know if the reaction occurs correctly or not. 

Controls in the PCR reaction work as a quality assurance that leads us to a conclusion of the success or failure of the experiment and also explains which are the possible reactions the reaction includes while failing. 

It certainly sets parameters for a single kind of reaction to repetitive use and hence PCR controls have significant importance in the diagnostic industry. 

It looks a bit confusing right! Let it be there! and start understanding from the basics. The PCR is a sensitive technique, a minute change in any parameters leads to a fatal error in the results either false-positive or false-negative results. 

Here is the list of some such parameters 

  • The quality and quantity of ingredients 
  • Reagent preparation 
  • Amplification conditions 
  • PCR cycling conditions 
  • The quality of Taq DNA polymerase 
  • The time of PCR run 
  • The composition of PCR reaction buffer 
  • The quality and quantity of template DNA
  • Lab conditions

Diluting parameters or compromising conditions can result in misleading results. Such misleading results are, 

  • No amplification
  • Poor amplification 
  • Primer dimer formation. 

These are the three common results but we will get many other false results, for instance, if we forget to add template DNA but still get amplification, that’s a type of false result. 

What does it show? We have done something wrong, isn’t it! But we don’t know if it is wrong or not because we do not have the “reference” to compare it. One such reference is our DNA ladder in agarose gel electrophoresis

The DNA ladder or marker allows one to investigate the size of each DNA amplicon run on a gel, imagine a gel without a DNA ladder, we can interpret nothing right! Those references are our controls in PCR. 

Those are some pre-set parameters using which we will interpret our PCR results. The use of control gives us an idea about 

  • The performance of the test 
  • Help to evaluate reasons for false results 
  • Help troubleshooting
  • Help understanding how results are different from the standard
  • Provides optimization strength for next reactions or experimental setup. 

Using more controls will give more data for future optimizations meaning it helps us with running the experiments and provides data or results to make a decision on what to do and what not to do next.

Depending on the type of PCR and assay requirement ‘controls’ vary, we will discuss different types of controls used in conventional and RT PCR. 

Types of PCR Controls: 

PCR commonly has two types of controls; Positive and Negative control, The positive control can be sub-divided into internal control, external control and reaction controls. 

Negative control: 

This is often known as Negative amplification control or no template control. It’s one of the simplest and most common PCR controls. These controls usually lack the template DNA, meaning, while preparing the reaction, we do not add the template DNA. 

The final results show nothing in the negative control lane. However, it shows that none of the reagents have been contaminated. 

When adding H20 or nuclease-free water instead of the template to the negative control, it becomes more aggressive. No amplification shows that the nuclease-free water used to prepare the reaction is of good quality and pure. 

The use of negative control gives us data regarding, 

  • If reagents are contaminated or not 
  • If any foreign DNA, viral DNA or naked DNA is accidentally incorporated in the reaction or not. 
  •  If the nuclease-free water is of high quality or not. 
  •  How to set up Negative control? 

The negative control tube included Taq DNA polymerase, forward and reverse primers, PCR buffer, nuclease-free water and PCR enhancers (if required). 

  • What is not included in the Negative control? 

Usually, the negative control tube lacks “template DNA”. 

As we said, the negative control has great value in conventional PCR, quantitative PCR and RT-PCR. Whatever the platform is, the negative control should not have any nucleic acid amplification reaction. 

Note that, if we use a larger amount of primers, and the template region is very rigid for amplification, we may get a primer-dimer zone even in the negative control. Because it lacks templates, not primers. 

Sometimes spurious primer amplification may be seen, but it isn’t our target amplification. If it shows amplification (DNA band in a gel) of any size, confirm that any reagent or reagents got contaminated. In that case, it’s advisable to use a fresh batch of reagents. 

Positive Control: 

The positive control can be further divided into, 

  • True positive control 
  • Internal control 
  • Standard control 
  • External control 

Put simply, the Positive control shows amplification thereby providing information that ensures a positive amplification reaction. A reaction needs two types of positive controls as a native (or external) positive control and internal control.

A separate external positive control tube is prepared which includes all the ingredients such as Taq DNA polymerase, PCR reaction buffer, dNTP mix nuclease-free water and a specific template. 

Here, a previous amplicon (of the same size) or a commercially available standard template (included in the kit) is used as a positive control. It gives amplification much like our reactions. 

No amplification indicates something wrong with reaction preparation. Although it isn’t 100% reliable as sometimes it happens that we forget to add something only in the positive control tube. 

We can say, no amplification indicates the chances of false-positive results. Usually, ready-to-use kits for Thalassemia, Sickle cell anemia, pathogen direction and viral load detection kits provide such controls.   

Notedly, it provides some important information scientists usually omit, the amplicon size. When PCR conditions compromise, results show more than two amplicons or amplicons other than the target. 

In such cases, a correct positive control provides a reference value to validate our target amplicon.

  • How to set up positive control? 

A parallel reaction tube to our target samples is prepared including Taq DNA polymerase, dNTPs, Primers, dNTPs, template DNA and nuclease-water.  

  • What does it show? 

It gives us an idea if our polymerase is working efficiently or not and if all the ingredients are utilized correctly or not.  

Internal control: 

The internal control though is independent but more valuable than the native or external positive control. It is also known as an internal positive amplification control. 

This usually requires a multiplexing reaction to amplify another region along with our target. As a result, we observe two amplicons that specifically are our target and internal control. 

It checks the quality and accuracy of performance at a specific reaction level. For instance, we have 10 tubes in a reaction and wouldn’t observe amplification in tube 9, not an internal control or target. This shows an error in reaction preparation at a specific reaction level. 

It also gives information if each PCR component working accurately or not. However, as we are multiplexing two reactions, sometimes, it causes fatal errors, non-specific amplification and un-amplification.  

See the image below, 

The right side of the ladder shows some of the non-specific amplification bands and primer dimers with internal control.

Importantly these types of positive controls provide information related to reaction-specific or template-specific PCR inhibitors. 

On the downside, internal controls have serious drawbacks as multiplexing reaction makes reaction failure or false-positive results. 

  • How to set up internal control?

Another set of off-target primers are utilized in the reaction along with target-specific primers, Taq DNA polymerase, dNTPs, reaction buffer and nuclease-free water. 

  • What does it show? 

It gives us an idea if our polymerase is working efficiently or not and if all the ingredients are utilized correctly or not.  

Standard control: 

Standard control is nothing but the sample of the previous amplicon or any amplicons loaded along with the result samples on a gel. It actually can’t provide data regarding if the reaction occurred well or not rather it gives an idea about if there is something wrong with the gel preparation or not! 

Notwithstanding, it’s very important to include because sometimes it is just a simple gel preparation error that leads to us tons of repeat experiments, cost and frustration. 

External control: 

A positive or negative sample amplified parallel to the original reaction is known as external control. The external control is similar to our external positive control. It helps, investigating false-negative and false-positive results.  

Quantitative standard control: 

RT PCR and relevant assay usually need a quality control or standard predetermined control sample that forms a base for the standard curve for quantification. Commonly, the quantity of these controls is known and provided by the manufacturer.  

To understand the concept precisely let us take two examples one after another for PCR and qPCR. 

Suppose we have to amplify one 400 bp product in our PCR. It’s an ARMS PCR assay meaning it utilizes 4 different primer sets to amplify different alleles. If you wish to learn more on ARMS PCR, click the link. 

We have prepared a typical PCR reaction consisting of dNTP mix, Primer mix, Taq DNA polymerase, PCR reaction buffer, nuclease-free water and template, and putting reactions at standard PCR cycling conditions. 

Just look at the image below, showing the results of our Hypothetical situation. 

The original gel image for a sample along with some controls.

The first lane is our DNA ladder, the second lane consists of the PCR product which is the 300bp fragment- a normal sample. The third lane shows the results of the allele which is not amplified. 

The 4th lane is our positive control, I have a previous amplification product of this gene which I have used in the reaction. The fifth lane is the negative control showing nothing. The sixth lane shows the gel running (external control) which is a previously amplified gene product. 

The small band-like diffusion products are some primer dimers. While the ~1000 bp fragment in each lane is our internal control used to validate if every reagent is working fine or not. 

So this is the basic setup for any PCR reaction, all tubes and reaction parameters must be included.    

Let us take an example of a typical SARS-CoV 2 RT-PCR kit which comprises three types of controls. Positive, negative and internal control. 

As we said, negative control shows no amplification. It assure no contamination at any level of reaction preparation. 

The positive control consists of the nucleotide sequence similar to our target SARS CoV2  and shows amplification during the reaction. It also indicates that everything is working fine in the reaction. 

Internal controls are of two types, the kit manufacturer provides one as per their SOP. The endogenous internal controls which are typically housekeeping genes amplify along with the template showing that whether the starting template has sufficient quantity or not. 

The exogenous control is usually avoided to reduce the complexity of the assay. 

RT PCR gives us results as the standard curve needs negative control, positive quality control to determine results. 

Any background signal during reaction shows the presence of a contaminant. Besides providing quality assurance,  positive control provides a reference value to estimate the quantity of the template too. 

Control Ingredients Template DNAinformation
Negative controlTaq DNA polymerase, dNTPs, primer set, reaction buffer, nuclease-free water. Absent Validates reagents. Indicate contamination. 
Positive control Taq DNA polymerase, dNTPs, primer set, reaction buffer, nuclease-free water and templatePresent Validates Taq DNA polymerase activity. Provides reference value for the assay.  
Internal control Taq DNA polymerase, dNTPs, primer set, reaction buffer, nuclease-free water, template and internal control primersPresent Check sufficiency of template validates the activity of Taq. 

Wrapping up: 

PCR controls have significant importance in the PCR reaction. Though no reaction is carried out without controls, it has an unmatched value in the diagnostic industry. Companies are providing each control in the kit itself. 

SARS-CoV2 RT-PCR kit has negative, positive and internal control in the kit. 

I think this information makes you understand the concept of PCR controls, I have tried explaining everything and also used several pictures to let you understand it. 

Summary
Type of PCR Controls- Negative, Positive and Internal Controls
Article Name
Type of PCR Controls- Negative, Positive and Internal Controls
Description
Positive, Negative and Internal controls are common PCR control types employed as a reference to evaluate results. 
Author
Publisher Name
Genetic Education
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