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Molecular Biology

Prevention

The following review details the prevention of biological, amplified, and non-amplified nucleic acid contamination through workflow design, technique, and enzymatic controls

The Unidirectional Workflow

The most fundamental structural control against contamination is the establishment of a unidirectional (one-way) workflow. This physical separation prevents “carryover” - the accidental transfer of previously amplified DNA (amplicons) into new, unamplified patient samples

  • Physical Separation of Areas: The molecular laboratory must be divided into distinct workspaces, ideally in separate rooms. If separate rooms are not available, distinct “dead air” boxes or PCR workstations must be used
    • Area 1: Reagent Preparation (Clean Area): This is the sanctuary of the lab. Only preparation of Master Mix occurs here. No patient samples or DNA are ever introduced to this area
    • Area 2: Specimen Preparation/Extraction: Patient samples are processed, and nucleic acids are extracted here
    • Area 3: Amplification and Detection (Dirty Area): This is where PCR cycling and post-PCR analysis (gel electrophoresis, capillary electrophoresis) occur. This area contains billions of amplicons and is considered the source of highest contamination risk
  • Movement Protocols: Personnel and specimens must always move from Clean → Dirty (Reagent Prep → Specimen Prep → Amplification)
    • Laboratory scientists should never move from the Amplification area back to Reagent Preparation during the same shift without a thorough decontamination procedure (showering/changing clothes), though most labs simply prohibit this “backtracking” entirely
    • Paperwork and lab notebooks should ideally not travel backwards against the workflow

Dedicated Equipment & Consumables

To prevent cross-contamination between areas, equipment must be dedicated to specific zones. An item used in the “Post-PCR” room should never enter the “Reagent Prep” room

  • Pipettes: Each workspace must have its own set of color-coded or labeled pipettes. A pipette used to load a gel in Area 3 will have amplicon aerosols on it; bringing it to Area 1 guarantees contamination of the Master Mix
  • PPE (Personal Protective Equipment)
    • Lab Coats: Must be dedicated to specific rooms. “Clean” room coats should never be worn in “Dirty” rooms. Front-closing coats are standard, but back-closing gowns are preferred in reagent prep to prevent skin shedding from the chest/neck area falling into tubes
    • Gloves: Gloves must be changed immediately if splashed, frequently during protocols, and always when moving between different workflow areas. Powder-free gloves are mandatory, as powder can inhibit enzymes
  • Consumables
    • Aerosol Barrier Tips (Filter Tips): All molecular pipetting must use tips containing a hydrophobic filter. This prevents aerosols (micro-droplets of liquid created during pipetting) from contaminating the barrel of the pipette
    • Aliquotting Reagents: Reagents (primers, dNTPs, buffers) should be aliquotted into single-use volumes. If a stock tube is contaminated, the entire lot is lost. Single-use aliquots limit contamination to one reaction set

Technique & Handling

Even with perfect room design, poor laboratory scientist technique can introduce contamination. This applies to biological contamination (patient-to-patient) and environmental genomic contamination (technologist-to-sample)

  • Aerosol Minimization
    • Centrifuge tubes briefly (pulse spin) before opening to ensure liquid is at the bottom and not on the cap/rim, which reduces splashing
    • Open tubes carefully; avoid “popping” caps, which creates invisible aerosols
  • Pipetting Technique
    • Avoid touching the sides of the tube with the pipette shaft
    • Hold the pipette vertically to ensure precise measurement and prevent liquid from touching the filter
  • Surface Decontamination
    • Chemical: Surfaces should be cleaned before and after work with 10% Sodium Hypochlorite (bleach). Bleach chemically destroys DNA. However, bleach is corrosive; it must be followed by a rinse with 70% ethanol or DI water to remove residue and prevent pitting of stainless steel hoods
    • UV Irradiation: “Dead air” boxes and biological safety cabinets are often equipped with UV lights. UV light (at 254 nm) causes pyrimidine dimers (thymine dimers) to form in DNA, rendering it un-amplifiable

Enzymatic Control: The dUTP-UNG System

While physical controls are essential, they are not foolproof. The most robust method for preventing amplicon contamination (carryover) is a chemical substitution method incorporated into the PCR master mix

  • Principle: In the PCR Master Mix, the nucleotide dTTP (Thymine) is completely replaced with dUTP (Uracil)
  • Result: All amplicons generated by the lab will contain Uracil instead of Thymine. DNA is distinct from natural DNA (which contains Thymine)
  • Enzyme: The enzyme Uracil-N-Glycosylase (UNG), also known as Uracil-DNA Glycosylase (UDG), is added to the Master Mix
  • Mechanism
    1. At the very start of a PCR run, the mix is heated to 50°C for 2-5 minutes
    2. During this step, the UNG enzyme scans for any DNA containing Uracil (contaminating amplicons from previous runs)
    3. If found, UNG cleaves the glycosidic bond, degrading the contaminant DNA so it cannot serve as a template
    4. The reaction is then heated to 95°C for the denaturation step. This high heat destroys the UNG enzyme
    5. The Taq polymerase then amplifies the true patient target (which contains Thymine and was ignored by the UNG)
    • Note: This method works effectively for preventing amplicon contamination but does NOT prevent cross-contamination of biological patient samples (which contain natural Thymine).

Summary of Contaminant Types & Specific Prevention

Amplified Nucleic Acid (Carryover Contamination)

  • Definition: Previously amplified PCR products (amplicons) entering a new reaction. Because these are concentrated targets, this is the most severe risk in high-volume labs
  • Specific Prevention: Unidirectional workflow, dUTP-UNG enzymatic system, separate air handling, aerosol barrier tips

Biological Contamination (Cross-Contamination)

  • Definition: Genomic DNA/RNA from one patient sample entering another patient’s tube
  • Specific Prevention: Never have two patient tubes open at the same time. Change gloves between every sample. Use automated extraction systems when possible to reduce manual handling

Non-Amplified Nucleic Acid (Environmental/Genomic)

  • Definition: DNA or RNA from the laboratory scientist (hair, skin, sneezing) or microorganisms in the environment entering the reaction
  • Specific Prevention
    • RNase Free: For RNA work, RNases are ubiquitous (found on skin and hair). Wear gloves at all times, cover exposed skin, and use RNase removal sprays (e.g., RNaseZap) on surfaces
    • Positive Pressure: The Reagent Prep (Clean) room should ideally be under slight positive pressure compared to the hallway to prevent dust and outside spores from entering when the door opens
    • NTC (No Template Control): Always run a “Blank” or Negative Control containing water instead of DNA. If this amplifies, you have contamination in your reagents or environment