Nucleic Acid Quality and Quantity Tests

• Nucleic acid quality and quantity can be measured using a variety of techniques.

Electrophoresis

• Electrophoresis separates particles in a solution or matrix by applying an electric current.
• Useful for analyzing DNA and RNA quality by resolving a sample aliquot on agarose.
• An electric current propels charged biomolecules through a porous gel matrix, and the rate is affected by the charge, size, and shape of the molecules.
• Fluorescent dyes such as ethidium bromide and SybrGreen I and II are used, and silver stain.
• The appearance of DNA on agarose gels varies depending on the type of DNA isolated.

Spectrophotometry

• Spectrophotometry measures the concentration of an analyte in a solution by measuring light absorption.
• Nucleic acids absorb light at 260 nm because of the adenine residues.
• Beer-Lambert Law: A = εbc, where A is absorbance, ε is molar absorptivity, b is path length, and c is concentration.
• Molar absorptivity (€) is 50 L/mol-cm for dsDNA and 40 for RNA.
• Absorbance is directly proportional to the concentration of the nucleic acid in the sample.
• To obtain the concentration in ug/mL: 0.200 absorbance units x 50 ug/mL per absorbance units x 100 = 1,000 ug/mL
• Molar absorptivity (L/mol-cm), 50 for dsDNA, 40 for RNA
• At 260 nm, 1 optical density unit (or absorbance unit) = 50 mg/L (or 50 g/mL) of dsDNA & 40 pg/mL of RNA
• Absorbance reading x conversion factor x dilution factor

Fluorometry

• Fluorometry measures emitted fluorescent light.
• It is useful for detecting small amounts of analytes after using dyes.

Microfluidics

• Microfluidics uses systems that process or manipulate small amounts of fluid in channels that range from tens to hundreds of micrometers.
• Uses capillaries

UV Spectrometry

• Standard nucleic acid quantitation
• A nucleic acid sample is placed into a quartz cuvette, which is then placed inside the UV spectrometer.
• UV light is passed through the sample at a specified path length, and the absorbance of the sample is measured at specific wavelengths.
• Does not require additional reagents or incubation time.

Fluorometry (Fluorescent Spectroscopy)

• Measures fluorescence related to DNA concentration in association with DNA-specific fluorescent dyes
• Early methods: 3,5-diaminobenzoic acid 2HCI (DABA), combined with alpha-methylene aldehydes (deoxyribose) to yield a fluorescent product
• Modern methods use the DNA-specific dye Hoechst 33258, combined with adenine-thymine base pairs in the minor groove of the DNA double helix.
• Fluorometric determination of DNA concentration can go down to 200 ng DNA/mL.
• PicoGreen can detect down to 25 pg/mL concentrations.
• OliGreen can detect down to 100 pg/mL of ssDNA.
• SybrGreen II RNA gel stain is used for RNA, with a sensitivity of 2 ng/mL.

Considerations for Absorption and Fluorometry

• Absorption and fluorometry readings may not always agree because these methods recognize different targets.
• Single nucleotides do not bind to fluorescent dyes, but they can absorb ultraviolet light.
• Absorption measurements do not distinguish between DNA and RNA.
• Spectrophotometry is most used because the samples can be read directly without staining or mixing with dye.
• Fluorometry may be preferred for methods requiring accurate measurements of low amounts of DNA/RNA (in the range of 10 to 100 ng/mL).

NanoDrop Spectrophotometry

• Similar in principle to traditional spectrophotometry but with additional capabilities.
• Functions by combining fiber optic technology and natural surface tension properties.
• Special software enables analysis of signal from small quantities of sample.
• Displays the entire absorbance spectrum of the sample in graphical form, enabling detection of contaminants.
• Capable of determining a wide range of sample concentrations without requiring serial dilutions.

Microfluidics

• A sample is applied to a multi-well chip and then moves through microchannels across a detector.
• Instrument software generates images in electropherogram (peak) or gel (band) configurations.
• RNA integrity number: a quantification estimate for RNA, determined as a standard measure of RNA integrity.
• Uses a minimal volume of sample (as low as 1 uL) and can test multiple samples simultaneously.
• Useful for analysis of studies on small RNAs (microRNAs) in eukaryotes and gene expression in bacteria.

Estimating Nucleic Acid Purity

• Contamination is detected by reading the concentration over a range of wavelengths.
• Contamination is indicated by absorbance over background at any wavelength other than the A260 maxima of the nucleic acid.
• A260/A280 ratio is used to assess DNA purity:
  • A260/A280 DNA = 1.6 to 2.0 indicates that the DNA is suitable for use.
  • A260/A280 RNA = 2.1 to 2.3 indicates that RNA is present instead of DNA.

Common Contaminants and Their Wavelengths of Peak Absorbance

• 230 nm: Organic compounds
• 270 nm: Phenol
• 280 nm: Protein

• 330 nm: Particulate matter