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Molecular Pathology

DNA purification

Analyzing Nucleic Acid Purity


Author: Rodney E. Shackelford, D.O., Ph.D. (see Reviewers page)
Revised: 6 March 2012, last major update January 2012
Copyright: (c) 2012, PathologyOutlines.com, Inc.

General
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● Many different methods are used to analyze nucleic acid purity; each has specific advantages and disadvantages

Spectrophotometric Analysis (A260/280 method)
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● A260/280 method of determining nucleic acid purity is quick and inexpensive
● Theory: nucleic acids absorb significant ultraviolet (UV) light at 260 nm, with the degree of ultraviolet light absorption being directly related to the nucleic acid concentration, by the Beer Lambert Law (Wikipedia)
● The extinction coefficient for single-stranded DNA is 0.027 (ug/ml)-1 cm-1, for double-stranded DNA is 0.020 (ug/ml)-1 cm-1, for single-stranded RNA is 0.025 (ug/ml)-1 cm-1
● Based on absorption at 260 nm, an optical density gradient or “OD” of 1.0 corresponds to 50 ug/ml for double-stranded DNA
● Proteins strongly absorb UV light at 280 nm, mainly due to the amino acids tyrosine, tryptophan, cysteine; thus, the concentration of contaminating proteins within a nucleic acid sample can be calculated from the absorbance ratios at 260 and 280 nm (A260/280)
● A260/280: ~1.8 for pure DNA samples; ~2.0 for pure RNA samples
● The A260/280 falls with increasing protein contamination, is ~0.57 for pure proteins in solution without nucleic acids
● Nucleic acid samples should be free of phenol, which absorbs strongly at 270 nm
● Pure nucleic acids should have zero absorbance at 330 nm; absorption at 330 nm and above indicates visible light absorption by particulates in solution

Nucleic Acid Quantification with Fluorescent Dyes
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● Can quantify nucleic acids by measuring fluorescence intensity of dye-bound nucleic acids
● Useful to quantify relatively minute nucleic acid concentrations, helpful if sample has too many absorbing A260 contaminants
● Accuracy due to specificity of nucleic acid binding and fluorescence
● Method: DNA or RNA-binding dye is added to a nucleic acid solution; solution is loaded into agarose gel or a surface like a plastic wrap
● Nucleic acid samples of known concentration are added to unknown sample and nucleic acid concentration is estimated by comparing unknown to known samples
● Although somewhat subjective, this method works well for samples with low concentrations of nucleic acids
● This method can be made more exact using microplates or cuvettes measurements against a standard curve on a fluorescent photometer
● Dyes include ethidium bromide, SybrGreen 1, cyanine dyes, RiboGreen, PcoGreen, and DAPI

End of Molecular Pathology > DNA purification > Analyzing Nucleic Acid Purity


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