Chapter 25

Quantitative Estimation of DNA and RNA

Principle: The purine (adenine and guanine) and pyrimidine
(cytosine, thymine, and uracil) bases of DNA and RNA can be
estimated quantitatively and qualitatively using UV spectropho-
tometer, because the conjugated double bonds of nucleic acids
tend to absorb ultraviolet light between 220 nm and 300 nm. On
solubilizing the DNA in citrate buffer and measuring its optical
density at 260:280 nm, the ratio of 1.85 indicates that the isolated
DNA is pure. The ratio changes to ~2.0 for RNA and decreases
below 1.85 for protein contaminations if any. Nomogram is the
scale which indicates the amount of both RNA and DNA in a
sample at the same time.

Combined Viva Voce for Experiment Determination of Melting

Temperature of DNA and Quantitative Estimation of DNA
and RNA:
Q1. What is meant by melting of DNA?
Q2. What is the principle of determining melting temperature of
DNA?
Q3. Which buffer is used for dissolving DNA?
Q4. What is the most abundant form of DNA in nature?
Q5. _____________temperature range is used for determining
melting temperature of DNA.
Q6. What is the absorbance ratio of pure DNA at 260:280 nm?
Q7. The value of 2.0 obtained for 260:280 ratio at UV is
for_____________
Q8. The value less than 1.85 indicates_____________contamina-
tion in isolated DNA sample.
Q9. The scale used for estimation of RNA and DNA in a sample at
a time is called as_____________
Q10. Which wavelength is used for UV estimation of DNA and
RNA?

Aakanchha Jain et al. Basic Techniques in Biochemistry, Microbiology and Molecular Biology: Principles and Techniques,
Springer Protocols Handbooks, https://doi.org/10.1007/978-1-4939-9861-6_25,
© Springer Science+Business Media, LLC, part of Springer Nature 2020

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