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Tuesday, March 5, 2013

DNA Quantification and Quality Analysis


After isolation of DNA, quantification and analysis of quality are necessary to ascertain the approximate quantity of DNA obtained and the suitability of DNA sample for further analysis. This is important for many applications including digestion of DNA by restriction enzymes or PCR amplification of target DNA. The most commonly used methodologies for quantifying the amount of nucleic acid in a preparation are: (i) gel electrophoresis; and (ii) spectrophotometric analysis. If the sample amount is less, the former method is usually preferred.

A. Agarose Gel Electrophoresis for DNA Quantification and Quality Analysis


This method of quantification is based on the ethidium bromide fluorescent staining of DNA. Ethidium bromide is a fluorescent dye, which intercalates between the stacked bases. The fluorescent yield of the dye:DNA complex is much greater than the unbound dye. UV irradiation at 254nm is absorbed by the DNA and transmitted to the dye and the bound dye itself absorbs radiation at 302nm and 366nm. This energy is retransmitted at 590nm, the reddish-orange region of the visible spectrum. In case of plant genomic DNA, the nucleic acids are electrophoretically separated on a 0.7-0.8% agarose gel containing ethidium bromide at a final concentration of 0.5 ug/ml. The quantity of DNA can be estimated by comparing the fluorescent yield of the samples with a series of standards, for instance, lambda DNA at varying known concentrations. This provides a very rapid and sensitive means of estimating the nucleic acid concentration. A large number of samples with as little as 1-5ng of DNA can be quantified. Besides quantification, it also allows provides the advantage of analyzing the quality of the DNA preparation. Native DNA, which migrates as a tight band of high molecular weight (> or = 40 kb), presence of RNA, and degraded/sheared DNA, if any, can be visually identified on the gel.

Procedure

Prepare a 0.8% agarose gel.

Add 1 ul of 6X gel loading dye to 2-3 ul of each DNA sample before loading the wells of the gel. Addition of dye allows us to note the extent to which the samples might have migrated during electrophoresis, so that it can be halted at an appropriate stage.

Load at least 1 or 2 wells with uncut, good quality lamba DNA or any previously quantified DNA samples (50ng and 100ng) as molecular weight standards.

Run the submarine electrophoretic gel at 70V till the dye has migrated one-third of the distance in the gel.

DNA can be visualized using a UV transilluminator and quantified in comparison with the fluorescent yield of the standards.

Note: For SSR and RAPD analysis, it is more important to have good quality DNA samples (unsheared/undegraded DNA), than high quantities of DNA. In contrast, RFLP analysis requires larger quantities of DNA, since the technique is not PCR-based.

B. Spectrophotometric Determination

Analysis of UV absorption by the nucleotides provides a simple and accurate estimation of the concentration of nucleic acids in a sample. Purines and pyrmidines in nucleic acid show absorption maxima around 260nm (eg., dATP: 259nm; dCTP: 272nm; dTTP: 247nm) if the DNA sample is pure without significant contamination from proteins or organic solvents. The ratio of OD260/OD280 should be determined to assess the purity of the sample. This method is however limited by the quantity of DNA and the purity of the preparation. Accurate analysis of the DNA preparation may be impeded by the presence of impurities in the sample or if the amount of DNA is too little. In the estimation of total genomic DNA, for example, the presence of RNA, sheared DNA etc. could interfere with the accurate estimation of total high molecular weight genomic DNA.

Procedure

Take 1 ml TE buffer in a cuvette and calibrate the spectrophotometer at 260nm as well as 280nm.

Add 10 ul of each DNA sample to 900ul TE (Tris-EDTA buffer) and mix well.

Use TE buffer as a blank in the other cuvette of the spectrophotometer.

Note the OD260 and OD280 values on spectrophotometer.

Calculate the OD260/OD280 ratio.

Comments:

A ratio between 1.8-2.0 denotes that the absorption in the UV range is due to nucleic acids.

A ratio lower than 1.8 indicates the presence of proteins and/or other UV absorbers.

A ratio higher than 2.0 indicates that the samples may be contaminated with chloroform or phenol. In either case (<1.8 or >2.0) it is advisable to re-precipitate the DNA.

The amount of DNA can be quantified using the formula:

DNA concentration (ug/ml) = (OD260 x 100 (dilution factor) x 50 ug/ml ) / 1000

Spectrophotomteric Conversions for Nucleic Acids:

1 A 260 of ds DNA = 50 ug/ml

1 A 260 of ss oligonucleotides = 33 ug/ml

1 A 260 of ss RNA = 40 ug/ml

Reference

Hoisington, D. Khairallah, M. and Gonzalez-de-Leon, D. (1994). Laboratory Protocols: CIMMYT Applied Biotechnology Center. Second Edition, Mexico, D.F.: CIMMYT.