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Tuesday, May 8, 2012

Nucleic Acid Quantification - DNA/RNA Quantification and Analysis


Nucleic Acid Quantification

Nucleic acids are quantified to check the concentration and purity of DNA/RNA present in the solution mixture.it is important to know the concentration and purity of the nucleic acid for the use in further applications like PCR, restriction digestion etc. Spectrophotometric analysis is the most commonly used method of quantifying DNA, agarose gel electrophoresis can also be used to analyse the DNA sample for purity.

 

Spectrophotometric Mehtod

Nucleic acids absorbs UV light in the wave length of 260nm, a solution containing nucleic acid to be tested is exposed to UV light and the absorbance is measured, the more light it absorbs the more nucleic acid your test solution contains.calculations are made according to the Beer Lambert's Law.

NanoDrop is a UV-Vis Spectrophotometer device from thermo scientific, which can quantify nucleic acid from micro volumes of 0.5µL – 2.0µL. Features of this product involves

Direct, easy measurements in less than 5 seconds – just pipette & wipe

Measures DNA, RNA (A260) and Protein (A280) concentrations and sample purity (260/280 ratio)

Large concentration range (2 ng/µL – 15,000 ng/µL dsDNA) without dilutions.

The latest NanoDrop series are NanoDrop 2000/2000c NanoDrop 8000, NanoDrop Lite, etc



A NanoDrop Device


Nanodrop


The 260/280 ratio tells us the purity of the sample analysed, Pure DNA sample gives a 260/280 ratio ~ 1.8 and for pure RNA 260/280 ratio is ~ 2.

Similarly, absorbance at 230 nm is accepted as being the result of other contamination; therefore the ratio of A260/A230 is frequently also calculated.. The 260/230 values for “pure” nucleic acid are often higher than the respective 260/280 values. Expected 260/230 values are commonly in the range of 2.0-2.2. 


Residual chemical contamination from nucleic acids extraction procedures may result an overestimation of the nucleic acid concentration and/or negatively influence downstream analysis. Example spectra for 4 common extraction reagents which, if not properly cleaned up, will affect sample purity. 



Image Source : Nanodrop Technical Literature

Checking Contaminants in the Extracted sample

  • A low 260/230 ratio indicates the contaminants absorbing at 230nm or less.
  • A low 260/280 ratio indicates the contaminants absorbing at 280nm or less.
  • A shift in the wavelength trough is indicative of contaminants absorbing at low wavelengths.
  • The wavelength of the sample peak should be at 260nm if contaminants are present the peak may shift.

Nanodrop peak shift


If the sample is contaminated with proteins or other organic compounds, the 260/280 ratio will vary from the above mentioned values.


Agarose Gel Electrophoresis
 


gel electrophoresis
 

It is a separation method used to separate nucleic acids based on their size under the influence of electric current. Since Nucleic acids are negatively charged, on applying electric field they move from cathode to anode. Depending upon the size of the nucleic acid to be analyzed suitable gel concentration can be made which act as a sieve to separate nucleic acids based on their size.

Purity of the sample can be analyzed using this method. Some time contaminating DNA/RNA fragments can be removed using this method. After running the sample on gel the band of interest can be spliced out and gel extraction can be done to purify it.