A wide variety of DNA and RNA sample types and extraction methods can introduce inhibitors to enzymatic reactions. Inhibitors, if not properly removed, can negatively affect many types of enzymatic reactions including reverse transcription, end repair, A-Tailing, adapter ligation, and PCR. This bulletin outlines important considerations for isolation and purification of DNA and RNA before library preparation with Illumina library preparation kits.
Which inhibitors are of concern?
Some inhibitors, or contaminants, are inherent to sample type or sample source, such as hemoglobin in blood or humic/fulvic acid in plant samples. Others are introduced through the sample treatment or extraction method such as EDTA, heparin, or phenol:chloroform.
Inhibitors can prevent the enzyme from binding to the target substrate (i.e., proteins coating DNA/RNA and thus preventing enzyme binding), reduce enzyme function, or degrade the enzyme itself (e.g., proteinases, detergents, phenol, and pH).
What if I suspect or detect contaminants in my DNA/RNA samples?
The best way to prevent enzyme inhibition or impaired performance is to couple careful sample handling with extraction protocols optimized to efficiently purify inhibitor-free nucleic acids. The final pH of the sample after eluting or resuspending nucleic acids should be within a range of 7.0–8.5.
Illumina recommends UV spectrophotometry for purity assessment, and fluorometric based methods such as Qubit or Pico/RiboGreen for nucleic acid quantitation. The most common method to assess the purity of nucleic acids in solution is to measure the 260/280 and 260/230 ratios by UV spectrophotometry:
What inhibitors are found in different sample types?
|Sample Source Inhibitors||Inhibitory Effect||Likely Source||Methods to Minimize Inhibition|
|Polysaccharides||Template blocking||Plants||High salt precipitation, CTAB Buffer, Chloroform extraction, Pectinase, Cellulase, Hemicellulase, α-amylase digestion|
|Proteins||Template blocking||Skin, Connective tissue, BSA, Immunoglobins||Use SDS, CTAB or Guanidinium buffers, Proteinase K, silica-based purification|
|Fats||Template blocking||Adipose Tissue; Glycerol||Upase or Hexane treatment and Chloroform extraction, silica- based purification|
|Bile Salts||Template blocking||Feces, Stool||Wash with 70% Ethanol or use silica-based purification|
|Collagen||Template blocking||Skin, Connective Tissue||Use SDS, CTAB or Guanidinium buffers, Proteinase K, silica-based purification|
|Heme||Competition with MgCl2||Blood||Wash with 70% Ethanol or use silica-based purification|
|Humic Acid||Chelation of metal ions||Soil, Plant Material||Wash with 70% Ethanol or use silica-based purification|
|Melanin and Eumelanin||Enzyme binding/ Template blocking||Hair, Skin||Wash with 70% Ethanol or use silica-based purification|
|Myoglobin||Chelation of metal ions||Muscle Tissue||Wash with 70% Ethanol or use silica-based purification|
|Complex Polysaccharides||Template blocking||Feces, Plant Material||High salt precipitation, CTAB Buffer, Chloroform extraction, Pectinase, Cellulase, Hemicellulase, α-amylase digestion|
|Proteinases||Template blocking||Milk||Only use carriers that do not serve as template or block the template such as linear acrylamide, N- or P-carriers|
|Calcium Ions||Competition with MgCl2||Milk, Bone||Wash with 70% Ethanol or use silica-based purification|
|Urea||Enzyme denaturation||Urine||Wash with 70% Ethanol or use silica-based purification|
|Hemoglobin, Lactoferrin||Competition with MgCl2||Blood||Wash with 70% Ethanol or use silica-based purification|
|Immunoglobin G (IgG)||Template blocking||Blood||Use SDS, CTAB or Guanidinium buffers, Proteinase K, silica-based purification|
|Indigo Dye; Tannic Acids||Template blocking||Specific Plants||Wash with 70% Ethanol or use silica-based purification|
What inhibitors are found in different samples treatments/extraction methods?
|Treatment/Extraction Method Carryover||Inhibitory Effect||Likely Source||Methods to Minimize Inhibition|
|EDTA||Chelation of metal ions||TE buffer||Reduce EDTA concentration in TE buffer or simply use Tris-HCl (10mM) or nuclease free water as elution buffer|
|Alcohols||Enzyme denaturation||Ethanol, isopropanol, isoamyl alcohol||Dry pellet and resuspend or use silica-based purification|
|Excess Salts||Template Blocking||KC, NaCl, CsCl, NaAc||Wash with 70% ethanol or use silica-based purification|
|Chaotropic Salts||Enzyme denaturation||Guanidinium chloride; Magnesium chloride, Urea||Wash with 70% ethanol or use silica-based purification|
|Phenol:Chloroform||Enzyme denaturation||Organic carryover||Use PVP, PVP/ammonium acetate, incorporation of 1.2% citric acid at the DNA extraction step|
|Detergents/DDT||Enzyme denaturation||Sodium deoxycholate, SDS, Tween 20, Triton X-100||Wash with 70% ethanol|
|Proteases||Protein degradation||Proteinase K||Phenol:chloroform ext followed by silica-based purification|
|Nucleases||Template degradation||Restriction enzymes, Micrococcal nuclease, S1||Use B-ME, EGTA, or SDS during protein precipitation|
|Exogenous DNA/RNA||Template competition||Carryover||DNAse I for DNA removal; RNAse A for RNA, RNA:DNA hybrid removal|
|Carriers||Template competition/blocking||RNA, Heparin, Glycogen||Only use carriers that do not serve as template or block the template such as linear acrylamide, N- or P-carriers|
|Agarose||Template blocking||Gel extractions||Use spin column with chaotropic salt buffer; dialysis|
|Excess Metal Ions||Reduce oligo specificity||Mg++ from PCR buffer||Dialysis against PBS (pH 7.4), phenol:chloroform extraction followed by EtOH precipitation|
How do I remove contaminants from my DNA/RNA samples?
If the sample has contaminants that affect downstream enzymatic reactions, additional purification steps such as a filter-based spin column repurification may help in the removal of contaminants and/or reconcentration of the sample.
Note that this list does not include all possible inhibitors of enzymatic reactions, but rather the most common contaminants that originate from sample source or sample treatment/extraction method.