2 Nucleic Acid Extraction

• UV absorbance at different wavelengths can indicate contamination in nucleic acid preparation

  • Strong A280 indicates contaminants such as proteins
  • Strong A230 may indicate contamination of organic compounds, ethanol, or salts
  • Strong A270 and A275 may indicate phenol contamination
• If the production yield is lower than expected but no degradation occurred, possible causes are:

  • Low quality of tissue or cell sample
  • Incomplete lysis
  • Incorrect extraction solutions
  • Incorrect elution solution
• RNase is heat-stable and very difficult to inactivate.
• In RNA degradation, storage temperature should be:

  • -20°C for short-term storage
  • -80°C for long term storage
• Improper storage temperature can lead to DNA degradation

  • 4°C for short-term storage
  • -20°C or -80°C for long term storage
• Large DNA molecules such as genomic DNA can break easily
• Acidic conditions can render DNA hydrolysis

  • 10mM Tris buffer (pH 8-9) → typically used for DNA
  • EDTA → commonly included in the storage solution to inhibit DNase activity
• All procedures involving phenol-chloroform are performed in a chemical fume hood
• Chloroform is a skin and eye irritant and a suspected carcinogen
• Phenol exposure through the skin can cause severe burns and systemic effects
• Visual observation of impurities:

  • Protein bands are not visible by ethidium bromide staining
  • Retention of nucleic acid in the well or a smeared band can indicate protein contamination
  • Nuclease activity is suspected when the band is smeared or faint
  • A smeared DNA band can also result from too much salt in the sample
• Quantification of nucleic acid can be done by gel densitometry, which compares the band intensity of the target nucleic acid to those of DNA standards
• SYBR Green is a less toxic stain with higher sensitivity; it is used as an alternative and requires a special optical filter for detection.
• Ethidium bromide is a stain that emits visible light upon excitation by UV light; it is toxic because it's an intercalating agent; mutagenic
• In purity analysis, nucleic acid bands can be visualized by a nucleic acid stain
• The principle of Gel electrophoresis is based on the electric current moves negatively charged nucleic acid from the negative end toward the positive end
• Gel electrophoresis is a method for separation of nucleic acid on a porous gel matrix based on their size and charge
• Modern method of fluorometry: RNA

  • SybrGreen II
• Modern method of fluorometry: DNA

  • DNA-specific dye Hoechst 33258 → combines with adenine-thymine pairs
  • PicoGreen → more sensitive, detects even if DNA is in picograms
  • Oligreen → binds to short pieces of single stranded DNA
• Early method of fluorometry uses 3,5-diaminobenzoic acid 2HCl (DABA) that combines with deoxyribose
• Fluorometry or fluorescent spectroscopy, measures fluorescence related to DNA concentration in association with DNA-specific fluorescent dyes
• At 260 nm nucleic acids are absorbed. For protein, it would be absorbed at 280 nm.

  We can determine if sample is contaminated with protein by computing the absorbance at 260 over 280 nm.
• For other contaminants such as chaotropic salts, the A260/A230 ratio is used since A230 is near the absorbance maxima of these impurities; Ratio should be >2.0
• In general, samples with good purity are:

  • DNA sample with an A260/A280 ratio of 1.6-1.9
  • RNA sample with an A260/A280 ratio >2.0
• Protein contamination in a nucleic acid sample can be indicated by the ratio of UV absorbance at 260 nm and 280 nm.
• Nucleic acid concentration can be estimated by measuring UV absorbance at 260 nm
• UV spectrophotometry and gel electrophoresis is commonly conducted in nucleic acid analysis
• Gel electrophoresis is the movement of molecules by an electric current; it dissolves nucleic acids on a gel matrix based on size and charge; gel and capillary electrophoresis
• Fluorometry uses fluorescent dye and spectrophotometer
• UV spectrophotometry measures the amount of light absorbed by nucleic acid
• After extraction, it is good practice to conduct nucleic acid analysis especially when the downstream applications demand specific purity and quantity of nucleic acids.
• polyA tail is found on the 3’ end of mRNA and consists of multiple adenosine monophosphate; it is added after the transcription process and gives protection to the mRNA as it goes out of the nucleus
• Extraction of RNA uses Trizol to inactivate endogenous RNase
• In Total RNA extraction:

  • rRNA → most abundant; 85%
  • tRNA → 10-12%
  • mRNA → 3-5%
• In extraction of mtDNA, the mitochondria is isolated by centrifugation, as well as the total DNA
• Magnetic bead-based extraction utilizes small particles with a paramagnetic core coated by a surface that can bind nucleic acid; it is common in automated nucleic extraction systems
• Solid-phase extraction is like a filter, no need for alcohol precipitation because end product is automatically purified
• The principle of Column-based extraction is selective binding of nucleic acid to a solid matrix in a column as a cell lysate is applied to the column
• In alcohol precipitation:

  • Upon addition of alcohol, the nucleic acid will precipitate at the bottom of the tube, and the upper part is removed.
  • Ethanol – to remove salts
  • Isopropanol – to precipitate nucleic acids
• After liquid-phase extraction, removal of residual solvents and salts from nucleic acid in solution is done by alcohol precipitation.

  • Isopropanol is used instead of ethanol when the sample volume is large.