MIDTERM LEC: MEASUREMENT OF NUCLEIC ACID QUALITY & QUANTITY Flashcards
WHAT ARE THE 4 METHODS MEASUREMENT OF THE QUALITY & QUANTITY OF DNA & RNA???
ELECTROPHORESIS
SPECTROPHOTOMETRY
FLUOROMETRY
MICROFLUIDICS
Separation of
particles through a solution or
matrix under the force of an
electric current
ELECTROPHORESIS
Technique that uses light absorption to measure the concentration of an analyte in solution
SPECTROPHOTOMETRY
Measurement of emitted fluorescent light
FLUOROMETRY
Science & technology of systems that process or manipulate small
amounts of fluid, using channels measuring from tens to hundreds of micrometers
MICROFLUIDICS
▪ DNA & RNA can be analyzed for quality (detection & size analysis) by resolving an aliquot of the isolated sample on an agarose gel
▪ Uses an electric current to propel charged biomolecules through a porous gel matrix at a rate that is the function of the charge, size, & shape of the molecules
ELECTROPHORESIS
Fluorescent dyes used in ELECTROPHORESIS
ethidium bromide, SybrGreen I & II, silver stain
Appearance of DNA on agarose gels depends on the type of DNA isolated
ELECTROPHORESIS
Nucleic acids absorb light at 260 nm through the adenine residues
SPECTROPHOTOMETRY
Beer-Lambert Law:
A = ∈bc
A = ∈bc
❖ A = absorbance
❖ ∈ = molar absorptivity (L/mol-cm), 50 for dsDNA, 40 for RNA
❖ b = path length (cm)
❖ c = concentration (mg/L)
Absorbance is _____ proportional to the concentration of the nucleic acid in the sample
directly
molar absorptivity for DNA???
50 mg/L or ug/mL
molar absorptivity for RNA???
40 mg/L or ug/mL
Using absorptivity, as a conversion factor from optical density to concentration:
At 260 nm, 1 optical density unit (or absorbance unit) = 50 mg/L (or 50 μg/mL) of dsDNA & 40 μg/mL of RNA
SPECTROPHOTOMETRY
determine concentration:
spectrophotometer reading in absorbance units x appropriate conversion factor
SPECTROPHOTOMETRY
If the DNA/RNA preparation require dilution before spectrophotometry, to determine concentration:
Absorbance reading x conversion factor x dilution factor
ESTIMATION OF PURITY OF NUCLEIC ACID
Detection of contaminants: _____
reading the concentration over a range of wavelength
ESTIMATION OF PURITY OF NUCLEIC ACID
Indication of contamination: _____
absorbance over background at any wavelength other than the A260 maxima of the nucleic acid
A260/A280 DNA= ______
1.6 to 2.00 times more than the absorbance at 280 nm
A260/A280 RNA=_____
2.0 to 2.3
A260/A280 = <1.6
contaminated
Most likely contaminant: protein (absorbs light at 280 nm through the aromatic tryptophan & tyrosine residues)
▪ Standard nucleic acid quantitation ▪ Nucleic acid sample is placed into quartz cuvette, which is then placed inside the UV spectrometer
▪ UV light passed through the sample at a specified path length, & the absorbance of the sample at specific wavelengths is measured
▪ Does not require additional reagents/incubation time
UV Spectrometry
Similar in principle with the previous, but has many additional capabilities ▪ Functions by combining fiber optic technology & natural surface tension properties
▪ Accompanied by special software to enable analysis of signal from small quantities of sample
▪ Displays the entire absorbance spectrum of the sample in graphical form 🡪 allows detection of contaminants
▪ Capable of determining a wide range of sample concentrations w/o requiring serial dilutions
NanoDrop Spectrophotometry
● Measures fluorescence related to DNA concentration in association with DNA-specific fluorescent dyes
● Early methods: 3,5-diaminobenzoic acid �HCl (DABA), combined with alpha-methylene aldehydes (deoxyribose) to yield a fluorescent product
● Modern methods: 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: down to 200 ng DNA/mL
FLUOROMETRY (FLUORESCENT SPECTROSCOPY)
Other DNA-specific dyes in FLUOROMETRY (FLUORESCENT SPECTROSCOPY)
PicoGreen = detection down to 25 pg/mL concentrations
OliGreen = detection down to 100 pg/mL of ssDNA
stain used for RNA IN FLUOROMETRY (FLUORESCENT SPECTROSCOPY)
SybrGreen II RNA gel stain is used (sensitivity = 2 ng/mL)
▪ 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 & RNA ▪ Deciding which instrument to use is at the discretion of the laboratory: ❖ Most use spectrophotometry because the samples can be read directly without staining or mixing with dye
❖ Methods requiring accurate measurements of low amounts of DNA/RNA (in the range of 10 to 100 ng/mL), fluorometry may be preferred
ABSORPTION & FLUOROMETRY READINGS MAY NOT ALWAYS AGREE
▪ Sample is applied to a multi-well chip & then moves through microchannels across a detector
▪ Instrument software generates images in electropherogram (peak) or gel (band) configurations
▪ RNA integrity number: quantification estimate for RNA, determined as a standard measure of RNA integrity
▪ Uses a minimal volume of sample (as low as 1 μL) & can test multiple samples simultaneously
▪ Useful for analysis of studies on small RNAs (microRNAs) in eukaryotes & gene expression in bacteria
MICROFLUIDICS
quantification estimate for RNA, determined as a standard measure of RNA integrity
RNA integrity number
