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How do neucleotide composition affect UV260:280 ratio during nucleic acid UV spectrophotometer reading?


As we know, one critical parameter to evaluate the quality of purified nucleic acid is UV 260:280 ratio. Based on the studies of nucleotide UV absorption, the UV 260:280 ratio for each nucleotide is as follows: Guanine: 1.15, Adenine: 4.50, Cytosine: 1.51, Uracil: 4.00 and Thymine: 1.47. The resultant 260:280 ratio for the nucleic acid being studied will be approximately equal to the weighted average of the 260:280 ratios for the four nucleotides present. So RNA will typically have a higher 260:280 ratio due to the higher ratio of uracil compared to that of thymine. Also, Nucleic acida in acidic solutions will under-present the 260/280 ratio by 0.2-0.3, while in a basic solution will over-present the 260/280 ratio by 0.2-0.3.

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