QUALITATIVE AND QUANTITATIVE ANALYSIS OF DNA BY SPECTROPHOTOMETRY

ABOUT AUTHORS:
Amar Nagesh Kumar¹, Reena jose², Praswetha Reddy³
1 Lecturer, Department of Biochemistry, SSR Medical College, Mauritius
2 M. Sc Biotechnology, Yogi Vemana University
3 M. Sc Biochemistry, Mahatma Gandhi P.G College, Guntur

OBJECTIVE:To estimate the given samples of DNA qualitatively and quantitatively and know its purity and concentration.

 BACKGROUND
Quantification of nucleic acids is commonly used in molecular Biology to determine the concentration of DNA and RNA present in the mixture As subsequent reaction or protocols using the nucleic acids sample of a required particular amount of optical performance. Both DNA and RNA exhibit strong absorbance of UV due to the presence of conjugated double bonds of the constant purine and pyramidine bases and these have characteristics of OD (optical density)of absorbance maximum at 260nm which is linearly related with the concentration  of the DNA in the solution up to the OD value of 2 . The spectroscopic method is used to check the purity of DNA. Proteins are the major contaminants in the nucleic acid extracts and these have the maximum absorbance at 280nm. Value less than 1.8 signifies the presence of proteins as impurities.

REFERENCE ID: PHARMATUTOR-ART-1134

MATERIALS & METHODS
Materials required
· SSC buffer(saline sodium citrate)pH 7
· Quarts cuvette
· Distilled water
· Tissue paper
· Conical flask
· Beaker
 UV spectrophotometer

REAGENT  PREPARATION:  
SSC BUFFER  20ml
* 0.3Mtrisodiumcitrate 1.7 gm dissolved in 20ml of distilled  water is 0.3 tri sodium citrate
* 3M sodium chloride
* 50 gm dissolved in 20ml of distil water is 3M sodium chloride

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PROCEDURE
Take 3ml of SSC buffer in the cuvette as blank .
Add 50μl of isolated DNA and make the volume up to 3ml by adding 2950µl ssc buffer.
Place the cuvettes in the spectrophotometer and measure the absorbance at 260nm and 280nm against blank .
The purity if the nucleic acids is determined by calculating the ratio of absorbance at  both  260 and 280nm by using the following formula. O.D _{260 nm}/ O.D _{280 nm.} Concentration of DNA (µg/ml)=O.D.₂₆₀X50Xdilution factor

For example:

Although O.D.₂₆₀ / O.D.₂₈₀   ratio of both the samples 1 and 2 is between 1.8 and 2.0 but sample A DNA Is better and pure because the ratio is close to 1.8.
While taking absorbance, the sample is diluted 100times, i.e. dilution factor is 100 times Conc. of DNA of sample1 = 0.233X50X100=1165µg/ml = 1.165µg/ml. 

Quantification of plasmid DNA:-
Isolated DNA was quantified by measuring the absorbance at 260 and at 280 nm. The ratio of absorbance 260/280 was used to determine the quality of the isolated DNA .The concentration was calculated using the following formula

Concentration of the DNA = OD₂₆₀ X 50 X dilution factor = mg of DNA /ml              

Quantified DNA was diluted to 150 ng/ml and used for polymerase chain

Polymerase chain reaction-
Polymerase chain reaction was performed using primers that were designed from the pUC19 vector sequence (Table). Primers were commercially obtained from Sigma–Aldrich (Sigma-Aldrich Inc. St. Louis, Missouri 63178, USA). All the primers designed were 20 base pairs or more in length with GC content in the range of 40-80%. Tm was calculated according to the formula Tm(C) = 2(A+T) + 4(G+C) -5. The annealing temperature for each pair of PCR primers was optimized experimentally.

PCR amplification was carried out with following reaction parameters

dNTPs                                                                                  200 µM

PCR reaction buffer                                                           1X

Magnesium chloride                                                         1.5 -2.5 mM

Primer (forward)                                                                 50 pmoles

Primer (reverse)                                                                  10 pmoles each

Templet DNA                                                                        150 ng

Taq DNA polymerase                                                         1U

The final reaction volume was 50 µl.

The cycling conditions used:

Initial denaturation                             94^oC, 2 min

Denaturation                                       94^oC, 45 sec

Annealing                                            54-62^oC, 30sec

Elongation                                           72^oC, 45 sec

Cycles                                                  35

Final elongation                                  72^oC, 10 min 

Agarose gel electrophoresis:
0.8 to1.5% agarose gels were prepared by dissolving the required quantity of agarose in 1X Tris-Acetate-EDTA (40mM tris-acetate. 1mM EDTA) electrophoresis buffer by heating in a microwave oven, followed by addition of ethidium bromide to a final concentration of 0.25 ug/ml. The agarose solution was poured into a gel tray containing a comb, allowed to cool and solidify, and then placed in electrophoresis tank and submerged in 1X TAE buffer. DNA samples were mixed with 6X gel loading buffer (0.25% bromophenol blue, 0.25% xylene cyanol 40% w/v sucrose). Samples were loaded on the gel along with DNA size standards. Horizontal electrophoresis was carried out at approximately 80-100V. The gel was photographed under UV light using the gel documentation system.

Ethanol precipitation-
Ice cold ethanol (100%) was added in a ratio of 1:5 (500 ul polled reaction mix and 2.5ml of ice cold ethanol). The mix was mixed properly and left at -80degC for 1 hour. After the incubation the DNA was precipitated by spinning at 12000rpm for 10min. The pellet is washed in cold 70% ethanol then after a further centrifugation step the ethanol is removed, and the nucleic acid pellet is allowed to dry before being resuspended in nuclease free water.

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RESULTS
The PCR primers were optimized for the annealing temperatures, MgCl₂ concentrations and template concentrations. The primers were standardized at a range (54degC- to 60degC) of annealing temperatures. The result was given in table (2). The amplification was good at 58degC for all the primers. The MgCl₂ concentrations of 1.5mM and 2.5mM were used to standardize the primers. The result was given in the table (3). All the products were amplified at both the MgCl₂ concentrations. Low MgCl₂ concentration was used in subsequent analysis. The template concentrations chosen for the amplification were 100 ng and 150 ng. The result was given in the table (4). After the amplification of the individual fragments with a volume of 50 ul each were pooled together and precipitated with 100% ethanol. After the ethanol precipitation the pellet was resuspended in 100 ul of the nuclease free water and 3 ul of the sample was loaded on the gel along with the reference molecular weight marker from MBI fermantas. The concentrated molecular weight marker and the reference marker were shown in the Figure (4). 

All the fragments intensity was good when compare to reference marker and the resolution between the marker bands between the home maid and commercial molecular weight markers were same.

Table1: The sequence of the primers used for the amplification of the different fragments in the molecular weight marker