DNA/RNA isolation considerations for Illumina Library Preparation Kits

12/21/18


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.