NUCLEIC ACID ANALYSIS MOLECULAR BIO.

Cabucana, Paul lester T. LAB

ANALYSIS OF NUCLEIC ACID QUANTITY AND PURITY

After extraction, it is a good practice to conduct a nucleic acid analysis, especially when downstream applications demand specific
purity and quantity of nucleic acid. There are a wide variety of options for the analysis of nucleic acid. Two commonly used methods
are: 
Spectrophotometric measurement - method that measures the amount of light absorbed by nucleic acid in samples. 
 Spectrophotometry is the most commonly used method for determining nucleic acid concentration.  
 Nucleic acid concentration can be estimated by measuring UV absorbance at 260 nm (A260) as the nitrogenous bases show
absorption maxima around 260 nm.  
 An estimation of the concentration of nucleic acid can be calculated from the following formula: 

 Spectrophotometric measurement is simple and fast and does not require complicated sample preparation. However,
spectrophotometry cannot discriminate between DNA and RNA. Some contaminants and proteins also interfere with A260 signals.
This detection method is relatively insensitive as it requires 5 µg/ml of dsDNA to observe an A260 of 0.1. 
Advantages 
 Simple and fast method 
 Easy sample preparations 
  
Disadvantages 
 No information about DNA and RNA cross-contamination or nucleic acid integrity 
 Interference of A260 from contaminants 
 Relatively insensitive detection 
GEL ELECTROPHORESIS

Gel electrophoresis - method that resolves nucleic acid on a gel matrix based on their size and charge. 
 Gel electrophoresis is a method for separation of nucleic acid on a porous gel matrix based on their size and charge. Electric
current moves negatively charged nucleic acid from the negative end toward the positive end. Smaller nucleic acid migrates
through the pores in gel medium faster than larger molecules. This allows separation of nucleic acid of different sizes. 
 It is important to ensure that the electrodes are oriented in the correct direction before you run gel electrophoresis. The electric
field will be reversed if the electrodes are reversed leading to nucleic acid migrating into a wrong direction. 
 A running time that is too long can cause smaller nucleic acid fragments to run off the gel. Select the proper gel percentage for
resolution of nucleic acid within the desired size range. 
 Nucleic acid bands, separated by gel electrophoresis, can be visualized by a nucleic acid stain. A commonly used
fluorescence stain is ethidium bromide, which emits visible light upon excitation by UV light. However, fluorescence stains with
much lower toxicity and higher sensitivity such, as SYBR Green, have been introduced as an alternative. 
 Quantification of nucleic acid can be done by gel densitometry. Since ethidium bromide stains dsDNA in a concentration-
dependent manner, the amount of nucleic acid can be estimated by comparing the band intensity of the target nucleic acid to
those of DNA standards. 
 Visual observation of characteristics of the nucleic acid bands after electrophoresis can also indicate the presence of impurities
in nucleic acid preparations. 
o Protein bands are not visible by ethidium bromide staining. However, retention of nucleic acid in the well or a
smeared band can indicate protein contamination. 
o Nuclease activity is suspected when the band is smeared or faint. 
o A smeared DNA band can also result from too much salt in the sample. 

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 NUCLEIC ACID ANALYSIS MOLECULAR BIO.
Cabucana, Paul lester T. LAB

 The picture below shows nucleic acid of optimal quality in lane 1. Lanes 2 and 3 show smeared bands that could be a result of
DNA degradation from nuclease contamination.

 Gel electrophoresis requires the preparation of a gel and the gel's exposure to a mutagen and UV light  in order to visualize
DNA fragments. Eye and face protection is absolutely required. However, the method provides some advantages over
spectrophotometric measurement. 
 
Advantages 
 Physical separation of nucleic acid from some contaminants. 
 Potential identification of unwanted nucleic acid. 
 Purification of nucleic acid from the gel for downstream applications. 
Disadvantages 
 A complicated technique required for separation of DNA larger than 20 kb. 
 Possible exposure to mutagenic agent if ethidium bromide is used as the stain. 
 Exposure to UV light upon band visualization. 
  
Note: Gels can be either prepared (homemade) or purchased (pre-cast). Prepared gels are less expensive, can be tailored to your
exact specifications, and can be prepared as needed. Pre-cast gels provide convenience and time savings, reduction of exposure to
toxic gel reagents, gel quality consistency, and reproducibility in data. 
SPECIAL PRECAUTIONS AND TROUBLESHOOTING
Organic extraction uses a mixture of phenol-chloroform that poses health hazards to laboratory personnel. Phenol exposure through the
skin can cause severe burns and systemic effects. Chloroform is a skin and eye irritant and a suspected carcinogen. To minimize
hazards from phenol-chloroform, the following practices should be adopted: 
 Perform all procedures involving phenol-chloroform in a chemical fume hood. 
 Wear PPE including approved eye and face protection, gloves, and protective clothing. 
 Acquire proper training for handling and disposal of the chemicals. 
 Refer to MSDS for additional safety and handling information. 
 Consult and follow your Laboratory SOPs 
SPECIAL PRECAUTIONS AND TROUBLESHOOTING : DNA DEGRADATION
Smeared DNA bands are one indication of potential DNA degradation. These are some other factors to consider to prevent DNA
degradation. 
 Acidic conditions can render DNA hydrolysis. A 10 mM Tris buffer (pH 8-9) is typically used for DNA storage where the
downstream applications allow. 
 Ethylenediaminetetraacetic acid (EDTA) is also commonly included in the storage solution to inhibit DNase
activity where the downstream applications allow. 
 Large DNA molecules such as genomic DNA can break easily. Physical manipulation such as excessive pipetting
and vortexing should be avoided. 
 Improper storage temperature can lead to DNA degradation. DNA can be stored at 4°C for short-term storage. For
long-term storage, DNA should be divided into small aliquots and stored at -20°C or -80°C to avoid repeated freeze-
thaw cycles. 

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 NUCLEIC ACID ANALYSIS MOLECULAR BIO.
Cabucana, Paul lester T. LAB

SPECIAL PRECAUTIONS AND TROUBLESHOOTING : RNA DEGRADATION

RNA is less stable than DNA and also susceptible to degradation that may occur from harsh lysis conditions, long periods of heating,
improper storage, or RNase contamination. To prevent degradation from temperature change, keep RNA in small aliquots at -20°C for
short-term storage, and at -80°C for long-term storage to preserve stability. 
RNase is heat-stable and very difficult to inactivate. Precautions must be taken to avoid degradation of RNA by RNase, such as: 
 Wearing gloves when handling RNA at all times. 
 Using RNase-free reagents and disposables such as tubes and barrier tips. 
 Adding additives such as RNase inhibitors in the lysis buffer when possible. 
 Decontaminating glassware and the work area. 
SPECIAL PRECAUTIONS AND TROUBLESHOOTING : LOW NUCLEIC ACID YIELDS
If the amount of nucleic acid is significantly lower than you expected and no degradation is observed, possible explanations are: 
 Low quality of tissue or cell sample. Proper treatment and storage of biological samples prior to extraction is essential for
preserving nucleic acid. 
 Incomplete lysis. Complete lysis is essential for successful release of nucleic acid from the cells. Insufficient lysis could be
due to an incorrect protocol or insufficient amount of lysis buffer. 
 Incorrect extraction solutions. When the buffers are not prepared correctly, nucleic acid may migrate to a different liquid
phase or may not bind to the solid phase. 
 Incorrect elution solution. In solid-phase extraction, the compositions and volume of elution buffer can greatly affect product
yields. 
SPECIAL PRECAUTIONS AND TROUBLESHOOTING : LOW PURITY
UV absorbance at different wavelengths can indicate contamination in nucleic acid preparation. 
 Strong A280 indicates contaminants such as proteins. This might be due to incomplete removal of proteins during extraction
especially when a large sample is processed without scaling up the extraction accordingly. 
 Strong A230 may indicate contamination of organic compounds, ethanol, or salts. An additional step of alcohol precipitation or
ethanol wash may be required. Residual ethanol can be removed by increasing the drying time of the nucleic acid pellet. 
 Strong A270 and A275 may indicate phenol contamination. This can be reduced by repeating alcohol precipitation or using a
column-based nucleic acid clean up kit. 
 

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