Nucleic Acid Extraction

Nucleic Acids Extraction: The Basics

There are a variety of different methods for performing nucleic acid extraction. Each can be divided into three steps: lysis, purification and recovery.

• Lysis is the disruption of tissues and cells in order to release the genetic material. Lysis can either be chemical, mechanical or enzymatic, and the method selected depends on the starting material and downstream application.
• Purification involves removing proteins, lipids, debris and other contaminants from the nucleic acids to isolating the DNA or RNA from the lysed material.
• Recovery of the purified nucleic acid material involves resuspension in a suitable medium, such as water or buffer solution, for preservation and so it is ready for digestion, electrophoresis, PCR, and any other desired downstream application.

Successful nucleic acid extraction requires sufficient amounts of reliable starting material. Efficient and reproducible sample preparation reagents and procedures are an important consideration for confident nucleic acid purification. This is especially true for difficult-to-lyse samples, such as when performing DNA extraction from tissue or environmental samples.

MP Bio offers a comprehensive selection of solutions to extract and purify nucleic acids from any source.

Nucleic Acid Extraction Methods

There are several effective methods for purifying DNA and RNA from biological samples¹. The choice of which methods to use depends on the sample type as well as the intended downstream uses for the extracted material.

• Guanidinium Thiocyanate-Phenol-Chloroform: A liquid-liquid extraction method that separates RNA from proteins, lipids and other nucleic acids. Samples are mixed with guanidinium thiocyanate, chloroform, phenol, and sodium acetate and centrifuged, separating the solution into an upper aqueous phase — containing the RNA — and lower organic phase.
• Cesium Chloride (CsCl)/ Ethidium bromide gradient centrifugation: High-speed centrifugation of CsCl solutions — an extremely dense salt — establishes a concentration gradient and density gradient with denser solutions (higher CsCl concentration) settling to the bottom of the tube. Ethidium bromide —— fluorescent under UV exposure — intercalates into the DNA within your sample, affecting the density and causing it to settle to the middle of the tube, while making it easy to visualize and isolate.
• Cetyltrimethylammonium Bromide (CTAB) extraction: Enables the extraction of DNA from plant tissues and food samples by separating polysaccharides from nucleic acids. CTAB concentration, salt concentration, and pH dictate the solubility of polysaccharides, nucleic acids, and proteins. Polyvinyl pyrrolidone is frequently added to this method to minimize binding of polyphenols to DNA following cell disruption. A common CTAB buffer recipe is: 2% CTAB, 1% polyvinyl pyrrolidone, 100 mM Tris-HCl, 1.4 M NacL, 20 mM EDTA.
• Alkaline extraction: Routinely used to isolate plasmid DNA from bacteria samples. Exposing harvested bacteria to highly alkaline solutions and sodium dodecyl sulfate removes proteins and most contaminants, including chromosomal DNA because it is often bound by proteins.
• Solid-phase extraction: Many commercial kits take advantage of the ability of nucleic acids to bind solid supports, such as silica. Spin columns or magnetic beads are used to bind the DNA or RNA, which are washed with alcohol to remove contaminants, then a solution is applied to solubilize and elute the nucleic acids.

Some nucleic acid extraction technologies, such as commercially available spin kits, are integrated, covering all steps of the procedure: lysis, purification, and recovery. Other sample types call for more involved and specialized procedures to retrieve a high yield of nucleic acids. Selecting the optimal lysis procedure for your sample ensures that your downstream applications will generate reliable results.

Assess Yield and Purity

A convenient way to measure the concentration and purity of your DNA and RNA is via optical density and absorbance using a spectrophotometer.

The concentration can be estimated by diluting the sample in water and measuring the absorbance at 260nm, adjusting the A260 measurement for turbidity (measured by absorbance at 320nm), multiplying by the dilution factor, and assuming:

• A260 of 1.0 = 50 µg/mL pure dsDNA
• A260 of 1.0 = 20-33 ng/µL of ssDNA
• A260 of 1.0 = 40 ng/µL of RNA

Concentration (µg/ml) = (A260 – A320) × dilution factor × 50µg/ml

Calculate the total yield by multiplying the concentration by the total volume of your concentrated sample.

Yield (µg) = concentration × total sample volume (ml)

Assess the purity of your sample by calculating the ratio of the absorbance at 260nm divided by the absorbance at 280nm, while correcting for turbidity (A320nm). High-quality DNA will have an A260/A280 ratio of 1.7–2.0.

Purity (A260/A280) = (A260 – A320) ÷ (A280 – A320)

Pro Tips for Common Problems

Extracting nucleic acids from some sample types can be technically challenging, and tough samples may require specialized procedures. For example, plants, heterogeneous mammalian tissues, and environmental samples can be tricky to work with, and highly variable in their composition. Below are suggested solutions for common problems in nucleic acid extraction.

Poor Quality - low molecular weight or degraded DNA

• Possible Cause
  • Starting sample or purified genomic DNA was not stored properly.
  • Starting sample was fixed with formalin or another fixative.
• Pro Tip
  • Nucleases may have degraded genomic DNA. Follow recommendations for storage and handling of your sample type.
  • Genomic DNA may be fragmented in the sample, making it infeasible to obtain high-molecular-weight DNA from this sample.
  • Do not store DNA in water for extended periods.

Low Yield

• Possible Cause
  • If the yield is low and purity/quality is good, the starting sample size was insufficient.
  • If yield and purity/quality are low, the starting sample was not stored properly.
  • Cells were not thoroughly lysed.
• Pro Tip
  • Amplify genomic DNA if your sample is limited.
  • Nucleases may have degraded genomic DNA. Follow recommendations for storage and handling of your sample type.
  • Follow homogenization/lysis recommendations for your sample type.

A260/A280 is <1.7 or >2.0

• Possible Cause
  • A260/A280 < 1.7: Proteins may be present.
  • A260/A280 > 1.9: RNA may be present.
• Pro Tip
  • Process starting sample according to recommended instructions to ensure thorough removal of proteins. Do not overfill the purification system. To remove protein contaminants, treat the sample with a protease or perform a phenol extraction with ethanol precipitation.
  • Ensure thorough washing to eliminate contamination in your final sample.
  • Some sample types contain high levels of RNA. RNA may not interfere with some applications.

DNA does not work in downstream application

• Possible Cause
  • Genomic DNA quality is poor.
  • If DNA quality is good: Contaminants may be present in the sample.
  • For silica purification using ethanol-based wash buffer: Residual ethanol remains in the sample.
• Pro Tip
  • Do not overload the purification system. This may result in poor yield and/or decreased purity of genomic DNA.
  • If organic solvents such as phenol were used, residual solvent may be present in the sample. Precipitate the DNA with salt and alcohol.
  • Thoroughly air dry the sample prior to elution.

Salt Contamination (Strong reading at 220-230 nm)

• Possible Cause
  • Guanidine salt (or other salt) entered the eluate due to the buffer or lysate contacting the upper column region.
• Pro Tip
  • Avoid touching the upper column area with the pipet tip; always pipet carefully onto the silica membrane.
  • Avoid transferring any foam that may have been present in the lysate; foam can enter into the cap area of the silica spin column.
  • Close the caps gently to avoid splashing the mixture into the upper cap area.

Overviews and case studies for challenging samples:

• Plants
• Skin Tissue
• Lung Tissue
• Wastewater
• Cassava Root
• Volcanic Ash Soil

Enhance Reproducibility with Automation

Automating nucleic acid extraction protocols facilitates increased reproducibility, efficiency, and throughput. Automation also negates the need for experienced operators, avoiding user error and potential issues with cross-contamination.

Automated platforms could be an efficient solution for users working with large sample numbers, as they provide the speed, ease-of-use, and reliability you need in your nucleic acid extractions.

References

1. Shin J.H. (2013) Nucleic Acid Extraction Techniques. In: Tang YW., Stratton C. (eds) Advanced Techniques in Diagnostic Microbiology. Springer, Boston, MA. https://doi.org/10.1007/978-1-4614-3970-7_11