# Aerosol Contamination in PCR: Causes and Prevention

## Introduction to Aerosol Contamination in PCR

The [polymerase chain reaction](/knowledge/molecular-biology/polymerase-chain-reaction) (PCR) is one of the most powerful and sensitive techniques in [molecular biology](/blog/careers/molecular-biology), capable of amplifying a single copy of a target DNA sequence into millions of detectable copies. This extraordinary sensitivity, however, is also the technique's greatest vulnerability. A single molecule of contaminating DNA introduced into a reaction can be amplified alongside the intended target, producing results that are misleading or entirely false. Among the various routes by which foreign DNA enters a PCR, aerosol contamination is the most insidious and the most difficult to control.

### What is Aerosol Contamination?

Aerosol contamination in PCR refers to the introduction of foreign DNA into a reaction mixture through microscopic droplets suspended in the air. These droplets, typically ranging from 0.1 to 100 micrometers in diameter, can contain DNA molecules from previous amplification reactions, from the laboratory environment, or from the person performing the experiment. When these droplets settle into an open reaction tube, a master mix, or onto laboratory surfaces that subsequently contact reaction components, they become a source of template DNA that the PCR will amplify alongside—or instead of—the intended target.

The term "aerosol" is used deliberately. Unlike bulk liquid contamination, where a visible drop of liquid transfers from one vessel to another, aerosol contamination is invisible. The droplets are too small to see, too light to reliably settle quickly, and capable of traveling surprising distances through the air. A tube opened on one bench can release a plume of DNA-containing microdroplets that drift across a room and settle into an open reaction vessel minutes later.

### Why It Matters in PCR

The consequences of aerosol contamination are severe because PCR is an exponential amplification process. Each cycle doubles the amount of target DNA, meaning that a single contaminating molecule can produce billions of copies by the end of a typical 30–40 cycle reaction. The contaminating DNA is often the product of a previous PCR—a situation known as amplicon carryover—and because it is already a complete amplicon, it amplifies with maximum efficiency. In fact, amplicon carryover is often more efficiently amplified than genomic DNA templates, which require an initial denaturation step to separate the two strands before primers can anneal.

For diagnostic applications, the stakes are particularly high. A false positive result caused by aerosol contamination can lead to misdiagnosis, unnecessary treatment, or failure to identify the true pathogen. In research settings, contamination wastes time, reagents, and effort, and can invalidate entire experiments. Understanding the sources, mechanisms, and prevention of aerosol contamination is therefore not an optional refinement but an essential competency for anyone working with PCR. For a broader overview of the technique itself, see [Polymerase Chain Reaction](/knowledge/molecular-biology/polymerase-chain-reaction).

## Sources of Aerosol Contamination

Aerosol contamination arises from multiple sources, and identifying them is the first step toward prevention. Some sources are inherent to the physical manipulation of liquids; others stem from the accumulation of amplification products in the laboratory environment.

### Pipetting and Vortexing

Pipetting is the single most common source of aerosol generation in the PCR laboratory. When a pipette tip is used to aspirate or dispense liquid, a thin film of liquid can remain on the outside of the tip. More importantly, the rapid expulsion of liquid from the tip creates shear forces that generate microdroplets. These droplets can be ejected from the tip as an aerosol, particularly when the final portion of the liquid is forcefully expelled—a practice known as "blow-out" that is common with serological pipettes but also occurs with micropipettors if the plunger is depressed too rapidly.

Vortexing is an equally potent source. When a tube containing liquid is vortexed, the vigorous agitation creates a vortex that throws droplets against the inside of the tube lid. When the lid is subsequently opened, these droplets are released into the air. The same principle applies to vigorous mixing by flicking or tapping the tube. Even the act of removing a pipette tip from the shaft of a micropipettor can generate a fine spray of droplets if residual liquid is present.

### Opening Tubes and Strips

The simple act of opening a tube after a [PCR reaction](/knowledge/molecular-biology/pcr-reaction) is a major source of aerosol contamination. During thermal cycling, the reaction mixture is heated to 94–98°C during the denaturation step. This heating causes evaporation and condensation, and the interior of the tube becomes coated with microscopic droplets of reaction fluid. When the tube is opened, these droplets are released as an aerosol. The problem is compounded when tubes are opened forcefully or when the lid is snapped open with a thumb, which acts like a pump, expelling the contents into the air.

This is why the post-amplification area is considered a "hot zone" in the PCR laboratory. Every tube that contains amplified product is a potential source of contamination, and the act of opening it, regardless of how careful the operator is, releases some amount of aerosolized DNA. Strip tubes and 96-well plates are particularly problematic because opening multiple wells simultaneously releases a larger cumulative aerosol.

### Amplicon Carryover from Previous Reactions

Amplicon carryover is the most common and most dangerous form of aerosol contamination. It occurs when the product of a previous [PCR reaction](/knowledge/molecular-biology/pcr-reaction)—the amplicon—contaminates a new reaction. Because amplicons are short, abundant, and already optimized for amplification by the same primers, they are the most efficient possible template for a new PCR.

The sources of amplicon carryover are numerous. A pipette tip used to load a gel with PCR product can contaminate the pipettor. Gloves that touched a tube containing amplified DNA can transfer that DNA to a bench surface, a tube rack, or the outside of a reagent bottle. The centrifuge used to spin down PCR tubes can become contaminated if a tube leaks during centrifugation. Even the act of discarding a used tip into a waste container can create a small aerosol if the tip contains residual PCR product. Over time, these small events accumulate, and the laboratory environment becomes progressively contaminated with amplicon DNA. This is why [PCR Specimen Contamination Is Rare](/knowledge/molecular-biology/pcr-specimen-contamination-is-rare) in well-run laboratories—not because the risk is low, but because rigorous protocols are in place to manage it.

## Mechanism of Contamination

Understanding the physical mechanism by which DNA-containing aerosols form and enter reactions is essential for designing effective prevention strategies.

### Formation of DNA-Containing Droplets

Aerosols form when mechanical energy is applied to a liquid surface. In the context of PCR, this energy comes from pipetting, vortexing, tube opening, or even walking across the laboratory floor. When a liquid film is disrupted, it breaks into droplets of varying sizes. Larger droplets (greater than 50 micrometers) fall quickly due to gravity, but smaller droplets (less than 10 micrometers) can remain suspended in the air for extended periods, behaving almost like a gas.

The DNA content of these droplets depends on the concentration of DNA in the source liquid. A PCR reaction that has completed 35 cycles typically contains approximately 10¹¹ to 10¹² copies of the amplicon per milliliter. Even a droplet of one nanoliter from such a reaction contains roughly 10⁵ to 10⁶ copies of the amplicon—far more than enough to contaminate any reaction it lands in. The problem is compounded by the fact that DNA is remarkably stable. It resists degradation at room temperature, survives drying, and can persist on surfaces for weeks or even months.

### Routes of Entry into PCR Reactions

Once DNA-containing aerosols are in the air, they can enter a PCR reaction through several routes. The most direct route is settling into an open tube. During reaction setup, tubes are often left open while reagents are added, providing a window of vulnerability. Aerosols can also contaminate the master mix itself. If a tube of master mix is opened in a contaminated environment, airborne DNA can settle into the tube, contaminating the entire batch of reactions prepared from that master mix.

A less obvious route is through contaminated surfaces and equipment. Aerosols settle onto bench surfaces, pipettors, tube racks, and gloves. When a pipettor is placed on a contaminated surface and then used to pipette reagents, the shaft of the pipettor can transfer contaminating DNA to the inside of a fresh tip. Similarly, a gloved hand that touches a contaminated surface and then touches the rim of a reaction tube can introduce DNA into the tube. This is why the distinction between "clean" and "dirty" hands is a fundamental rule in the PCR laboratory. For a more detailed discussion of how environmental factors contribute to contamination, see [Environmental Contamination PCR](/knowledge/molecular-biology/environmental-contamination-pcr).

## Impact on PCR Results

The consequences of aerosol contamination are not limited to the obvious problem of false positives. Contamination can affect PCR results in several ways, some of which are subtle and easily overlooked.

### False Positives and Misdiagnosis

The most direct consequence of aerosol contamination is the false positive result. In a diagnostic PCR, a false positive means that the test indicates the presence of a pathogen when the patient is not infected. This can lead to unnecessary antibiotic treatment, failure to identify the true cause of illness, and in the case of infectious disease outbreaks, inappropriate public health measures.

In research settings, false positives are equally damaging. A PCR designed to detect a rare transcript in a tissue sample may instead amplify contaminating DNA from a previous experiment, leading to the erroneous conclusion that the gene is expressed. The problem is particularly acute in experiments involving highly conserved genes or in studies of microbial communities, where contaminating DNA from the environment or from laboratory reagents can be mistaken for genuine biological signal. These issues are distinct from [Mycoplasma Contamination PCR](/knowledge/molecular-biology/mycoplasma-contamination-pcr), which involves contamination of cell cultures rather than PCR reactions themselves, but the consequences for experimental validity are similar.

### Effects on Quantitative PCR

Quantitative PCR (qPCR) is especially vulnerable to aerosol contamination because it relies on the measurement of amplification kinetics. In qPCR, the cycle threshold (Ct) value—the cycle at which fluorescence exceeds background—is inversely proportional to the starting quantity of template DNA. A contaminating amplicon, which is already a complete double-stranded DNA molecule, amplifies with maximum efficiency. This means that even a tiny amount of contaminating DNA can dramatically lower the Ct value, producing a result that suggests a much higher concentration of target than is actually present.

The effect is not always a simple shift in Ct values. If the contaminating DNA is present in some reactions but not others, it can increase the variance between replicate reactions, making the results statistically unreliable. In digital PCR, where the reaction is partitioned into thousands of individual micro-reactions, contamination can cause an increase in the number of positive partitions, leading to an overestimation of the target concentration. In all cases, the result is the same: the quantitative data no longer reflect the true amount of target in the original sample.

## Detection and Monitoring of Contamination

Detecting aerosol contamination requires a combination of negative controls and environmental monitoring. These measures do not prevent contamination, but they provide early warning that contamination is occurring, allowing corrective action before experimental results are compromised.

### No-Template Controls (NTCs)

The no-template control (NTC) is the most important control in any PCR experiment. An NTC contains all components of the reaction—buffer, dNTPs, primers, polymerase, and water—but no template DNA. If the NTC produces a positive signal, contamination is present. The NTC should be prepared last, after all sample reactions, using fresh reagents and a fresh pipette tip for each component.

The interpretation of NTC results requires care. In conventional PCR, the NTC should show no band on a gel after amplification. In qPCR, the NTC should show no amplification curve, or at most a signal that appears many cycles later than the weakest sample. A common mistake is to dismiss a weak NTC signal as "background." In reality, any amplification in the NTC indicates the presence of contaminating DNA, and the experiment should be repeated after decontamination. The NTC is also the first line of defense in monitoring the effectiveness of prevention strategies—if NTCs are consistently negative, the contamination controls are working.

### Surface and Air Sampling

Environmental monitoring involves testing surfaces and air in the laboratory for the presence of contaminating DNA. Surface sampling is performed by swabbing a defined area (typically 10 × 10 cm) with a sterile swab moistened with sterile water or buffer, then using the swab as the template in a PCR. Air sampling can be performed by placing open Petri dishes containing [PCR master mix](/knowledge/diagnostics/molecular/master-mix-components-in-pcr-roles-and-optimization-strategies) on the bench for a defined period (typically 30–60 minutes), then sealing the dishes and subjecting them to thermal cycling. Any amplification in these "settle plates" indicates airborne contamination.

These monitoring methods are most useful when performed on a regular schedule and when the results are recorded. A sudden increase in the frequency of positive surface samples or settle plates indicates that a contamination event has occurred and that the source should be identified. Monitoring is also useful for verifying that decontamination procedures have been effective. It is important to note that environmental monitoring detects the presence of amplifiable DNA, not necessarily the presence of viable organisms. This distinction is relevant because the goal of monitoring is to detect DNA contamination, not microbial contamination, which is a separate concern addressed by [Cell Line Contamination](/knowledge/molecular-biology/cell-line-contamination) protocols.

## Prevention Strategies

Prevention is the cornerstone of contamination control. The goal is to create a system of barriers and practices that minimize the generation of aerosols and prevent any aerosols that do form from reaching reaction components.

### Dedicated Areas and Equipment

Physical separation is the most effective strategy for preventing aerosol contamination. The PCR laboratory should be divided into at least three distinct areas: a reagent preparation area, a sample preparation area, and a post-amplification area. Each area should have its own dedicated equipment—pipettors, tube racks, centrifuges, and vortexers—that is never moved between areas.

The reagent preparation area is the "cleanest" area. Here, master mixes are prepared and aliquoted. No DNA samples, no PCR products, and no post-amplification materials should ever enter this area. The sample preparation area is where DNA is extracted from biological samples and added to the reaction tubes. The post-amplification area is where PCR products are analyzed by gel electrophoresis, sequencing, or other methods. This area is considered "dirty" and should be physically separated from the other areas, ideally in a different room.

Within each area, work should be performed in a laminar flow hood or PCR workstation. These hoods provide a filtered, particle-free environment and are particularly important in the reagent preparation area. The hood should be equipped with a UV lamp for decontamination between uses, and all work should be performed at least 15 cm away from the front edge of the hood to prevent airborne particles from entering the workspace.

### Aerosol-Resistant Pipette Tips

Aerosol-resistant pipette tips, also known as filtered tips or barrier tips, contain a porous filter that prevents aerosols and liquids from reaching the shaft of the pipettor. The filter is typically made of polyethylene or polypropylene and is positioned between the tip opening and the pipettor shaft. When liquid is aspirated, the filter allows air to pass but blocks liquid and aerosols. This prevents cross-contamination between samples and prevents the pipettor itself from becoming contaminated.

Filtered tips are not a substitute for good pipetting technique, but they are an essential additional barrier. They are particularly important when pipetting samples that may contain high concentrations of DNA, such as PCR products or plasmid preparations. It is important to note that filtered tips are single-use and should be discarded after each pipetting event. Reusing a filtered tip defeats the purpose of the filter and can introduce contamination.

### UV Irradiation and Chemical Decontamination

Ultraviolet (UV) irradiation is a standard method for decontaminating surfaces and equipment in the PCR laboratory. UV light at a wavelength of 254 nm damages DNA by inducing the formation of thymine dimers, which block amplification. UV irradiation is effective against dry DNA on surfaces but is less effective against DNA in liquid or in the presence of proteins, which can shield the DNA from UV damage.

For UV decontamination to be effective, the surface must be clean and free of organic material. The UV lamp should be positioned close to the surface (within 30 cm) and the exposure time should be at least 10–15 minutes. UV irradiation is most useful for decontaminating the interior of PCR workstations, pipettors, and other equipment that cannot be treated with liquid decontaminants.

Chemical decontamination is used for surfaces that can tolerate liquid treatment. Sodium hypochlorite (bleach) is the most effective chemical decontaminant for DNA. A 10% solution of household bleach (approximately 0.5% sodium hypochlorite) is sufficient to degrade DNA on contact. The surface should be wiped with the bleach solution, allowed to sit for at least 10 minutes, and then wiped again with water or 70% ethanol to remove residual bleach, which can inhibit PCR. Commercial DNA decontamination reagents, such as DNAZap or DNA Away, are also available and are effective for routine surface decontamination.

## Enzymatic and Chemical Controls

In addition to physical prevention, enzymatic and chemical methods can be used to inactivate contaminating DNA. These methods are particularly valuable for controlling amplicon carryover, the most common form of contamination.

### UDG and dUTP Incorporation

Uracil-DNA glycosylase (UDG), also known as uracil-N-glycosylase (UNG), is an enzyme that specifically recognizes uracil in DNA and removes it by cleaving the glycosidic bond between uracil and the deoxyribose sugar. This creates an abasic site that is not amplifiable by [DNA polymerase](/blog/guides/dna-polymerase). UDG is used in PCR through a strategy called the dUTP/UDG system.

In this system, deoxythymidine triphosphate (dTTP) is replaced with deoxyuridine triphosphate (dUTP) in the [PCR master mix](/knowledge/diagnostics/molecular/pcr-master-mix-components-optimization). During amplification, the polymerase incorporates uracil instead of thymine into the newly synthesized amplicons. The resulting amplicons contain uracil in place of thymine. Before the next round of PCR, the reaction mixture is treated with UDG, which degrades any uracil-containing DNA. Since the intended template (genomic DNA or cDNA) contains thymine, not uracil, it is not affected by UDG treatment. However, any contaminating amplicons from previous reactions, which contain uracil, are degraded and cannot serve as templates.

The typical protocol involves adding 1–2 units of UDG per 50 μL reaction and incubating at room temperature for 2–10 minutes before the initial denaturation step. The initial denaturation at 95°C for 10 minutes inactivates the UDG, allowing the subsequent amplification to proceed normally. The dUTP/UDG system is highly effective and is incorporated into many commercial PCR master mixes, particularly those designed for diagnostic applications.

### Other Decontamination Reagents

Several other reagents can be used to decontaminate surfaces and equipment. These include:

- **Hydrochloric acid (HCl):** A 0.1 M solution of HCl can degrade DNA on surfaces. It is effective but corrosive and should be used with caution.
- **Hydrogen peroxide:** A 3% solution of hydrogen peroxide can degrade DNA and is less corrosive than HCl.
- **IsoPsoralen:** This compound intercalates into double-stranded DNA and, when activated by long-wave UV light (365 nm), forms cross-links that block amplification. It is used to treat PCR products before they are opened in the laboratory, preventing them from serving as templates in subsequent reactions.

These reagents are useful for surface decontamination but are not suitable for treating reaction mixtures. The dUTP/UDG system remains the only widely used method for inactivating contaminating DNA within the reaction itself.

## Common Pitfalls and Troubleshooting

Even with rigorous protocols, contamination can occur. Recognizing common pitfalls and knowing how to troubleshoot them is essential for maintaining the integrity of PCR experiments.

### Pipetting Errors

Pipetting errors are the most common cause of contamination. The most frequent mistake is using the same pipette tip for multiple samples, which transfers DNA from one sample to the next. This is a particular risk when pipetting small volumes, where the difference between a "fresh tip" and a "reused tip" is not always obvious. Another common error is touching the pipette tip to the rim of the tube or to the bench surface, which can pick up contaminating DNA. Finally, pipetting too forcefully can generate aerosols that contaminate the surrounding area.

The solution is to use a fresh, filtered tip for every pipetting event, to pipette slowly and smoothly, and to avoid touching the tip to any surface other than the liquid being pipetted. When preparing a master mix, the mix should be pipetted into each reaction tube individually, rather than preparing a large volume and then dispensing it, as the latter approach increases the risk of aerosol generation.

### Inadequate Cleanup

Inadequate cleanup of the work area is another common pitfall. A bench that appears clean may still harbor DNA on its surface, particularly in areas that are not routinely wiped down, such as the edges of the bench, the handles of drawers, and the surfaces of equipment. The problem is compounded when cleaning is performed with water or 70% ethanol alone, which do not degrade DNA. Ethanol is a disinfectant but does not destroy DNA; it merely removes it from the surface if the surface is wiped thoroughly.

The solution is to use a dedicated DNA decontamination reagent, such as 10% bleach or a commercial product, and to clean all surfaces that come into contact with samples, reagents, or gloves. The cleaning should be performed at the beginning and end of each work session, and the cleaning solution should be allowed to remain on the surface for at least 10 minutes before being wiped off.

### Ignoring Controls

Ignoring or misinterpreting controls is perhaps the most dangerous pitfall. A no-template control that shows a positive signal is not a minor inconvenience; it is a clear indication that contamination is present and that the experimental results are unreliable. Yet students and even experienced researchers sometimes dismiss NTC signals as "primer dimers" or "background noise" and proceed with the experiment.

The solution is to treat every positive NTC as a contamination event. The experiment should be stopped, the source of contamination should be identified, and the decontamination procedures should be performed before the experiment is repeated. This may be time-consuming, but it is far less costly than publishing or acting on results that are contaminated. For a more detailed discussion of the factors that affect PCR reliability, see [Annealing Temperature Steel](/knowledge/molecular-biology/annealing-temperature-steel), which addresses how subtle changes in reaction conditions can affect specificity and sensitivity.

## Frequently Asked Questions

### What is aerosol contamination in PCR?

Aerosol contamination in PCR is the introduction of foreign DNA into a reaction through microscopic droplets suspended in the air. These droplets, which are invisible to the naked eye, can contain DNA from previous PCR reactions, from the laboratory environment, or from the person performing the experiment. When these droplets settle into an open reaction tube, a master mix, or onto surfaces that subsequently contact reaction components, they become a source of template DNA that the PCR amplifies, leading to false positive results.

### How does aerosol contamination occur?

Aerosol contamination occurs when mechanical energy is applied to a liquid containing DNA, causing the formation of microscopic droplets. Common sources include pipetting, vortexing, opening tubes after thermal cycling, and improper disposal of PCR products. The droplets can remain suspended in the air for extended periods and can travel significant distances before settling. Once settled, the DNA can be transferred to reaction components through contaminated surfaces, equipment, or gloves.

### What are the main sources of aerosol contamination?

The main sources are pipetting (particularly forceful expulsion of liquid), vortexing or vigorous mixing of tubes, opening tubes that contain amplified product, and amplicon carryover from previous reactions. The post-amplification area is a particularly rich source of contamination because it contains high concentrations of amplified DNA. Improper waste disposal, such as discarding tips or tubes containing PCR product in open containers, also contributes to environmental contamination.

### How can I prevent aerosol contamination in PCR?

Prevention requires a multi-layered approach: physical separation of pre- and post-amplification areas, use of dedicated equipment for each area, use of aerosol-resistant (filtered) pipette tips, UV irradiation and chemical decontamination of surfaces, and the use of enzymatic controls such as the dUTP/UDG system. Good pipetting technique, including slow and smooth pipetting and the use of a fresh tip for every pipetting event, is also essential.

### What is a no-template control (NTC) and why is it important?

A no-template control (NTC) is a reaction that contains all components of the PCR—buffer, dNTPs, primers, polymerase, and water—but no template DNA. It is prepared last, after all sample reactions. If the NTC produces a positive signal, contamination is present and the experimental results are unreliable. The NTC is the most important control in any PCR experiment because it provides a direct test for the presence of contaminating DNA.

### Can aerosol contamination affect quantitative PCR results?

Yes. In quantitative PCR (qPCR), contamination can lower the cycle threshold (Ct) value, producing results that suggest a higher concentration of target than is actually present. Contamination can also increase the variance between replicate reactions, making the results statistically unreliable. In digital PCR, contamination can increase the number of positive partitions, leading to an overestimation of the target concentration.

### What is the role of uracil-DNA glycosylase (UDG) in preventing contamination?

Uracil-DNA glycosylase (UDG) is an enzyme that removes uracil from DNA, creating abasic sites that cannot be amplified by [DNA polymerase](/blog/guides/dna-polymerase). In the dUTP/UDG system, dTTP is replaced with dUTP in the PCR master mix, so all newly synthesized amplicons contain uracil. Before the next round of PCR, the reaction is treated with UDG, which degrades any uracil-containing contaminating amplicons. The intended template, which contains thymine, is not affected. This system is highly effective for preventing amplicon carryover.

## Key Takeaways

- Aerosol contamination in PCR is the introduction of foreign DNA through microscopic airborne droplets, and it is the most common cause of false positive results.
- The primary sources of aerosol contamination are pipetting, vortexing, opening tubes after amplification, and amplicon carryover from previous reactions.
- Contamination can affect both conventional and quantitative PCR, leading to false positives, reduced sensitivity, and unreliable quantitative data.
- No-template controls (NTCs) are essential for detecting contamination, and any positive NTC signal should be treated as a contamination event requiring corrective action.
- Prevention requires physical separation of pre- and post-amplification areas, dedicated equipment, filtered pipette tips, UV irradiation, and chemical decontamination.
- The dUTP/UDG system is an enzymatic method that specifically degrades contaminating amplicons while leaving the intended template unaffected.
- Consistent monitoring, rigorous cleanup, and strict adherence to protocols are the most effective defenses against aerosol contamination.


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