Determine the (Tm) melting temperature of isolated DNA protocol

AIM

Determine melting temperature of the isolated DNA.

principle- 

When DNA is heated it denatures or melts i.e. the double stranded DNA separates into its single stranded components. When the temperature is decreased the strands re-associate to form the double stranded molecule (renaturation). The melting temperature (Tm) is defined as the temperature at

which half of the DNA strands have melted i.e half are in double-helical state and half are in the "random-coil" states. The melting temperature depends on the nucleotide composition of the DNA molecule and the length of the DNA. Higher Tm are associated with higher GC content, since GC base pairs are linked by three H-bonds while AT base pairs are linked by two H-bond. Hence

GC base pairs are stronger, requiring higher temperature for melting. The Tm can be used to calculate the GC content of the DNA.

Theory

DNA melting and reassociation can be monitored by measuring the absorbance at 260 nm. Double-stranded DNA has a lower absorbance, but when it is singlestranded, the unstacking of the bases leads to an enhancement of absorbance. This is called the hyperchromic effect. Therefore, the extent to which DNA is single-stranded or double-stranded can be determined by monitoring UV absorption. Temperature for midpoint of denaturation gives the Tm. By increasing temperature slowly and measuring absorbance at 260 nm as melting profile can be generated.

 Objective

1. To determine nucleic acid extraction efficiency and purity using UV spectrophotometry.

2. To measure the AT/CG ratio and the percentage of GC content of a DNA isolated from different sources (plasmid, plant and human) using the equation.

Materials

Chemicals

1. DNA Template (source; plasmid, human Genomic DNA, plant Genomic DNA)2. SSC buffer3. UV spectrophotometer and quartz cuvettes5.4.2 Equipments , Water bath2. Spectrophotometer UV Glassware, Quartz cuvette20 X SSC Buffer to make 1L use: 175.3 g NaCl 88.2 g Na Citrate dihydrate Dil HClDissolve in approximately 800 ml dH20. Adjust to pH 7.0 with dilute HCl. Bring up to a final volume of 1L and autoclave. Store at room temperature.

Experimental Protocol for the Characterization of DNA by Spectrophotometer Assay

1. Dissolve a small quantity of your extracted DNA in 3.0 ml of 0.1X SSC.

2. Turn on and blank a UV spectrophotometer at 220 nm (use 0.1X SSCas the blank). Determine the absorbance of your sample DNA at 230nm.

3. Change the wavelength to 230 nm, reblank the spectrophotometer and measure the absorbance of the sample at 230 nm.

4. Increment the wavelength by 10 nm and repeat blanking and measuring the absorbance until readings are taken through 300 nm.

5. Compute the absorbance ratio 260 nm to 280 nm. Pure DNA (without protein or RNA) will have a 260:280 absorbance ratio of 1.85. RNA will have a 260:280 ratio of 2.0.

6. Plot the absorbance spectrum of your sample and indicate the 260:280 ratio, as well as the amount of protein contamination on the graph.

Experimental Protocol for Melting Point Determination

1. Dissolve your DNA preparation in SSC to give a final concentration of approximately 20 µg DNA/ml.

2. Place the dissolved DNA in a quartz cuvette along with a second cuvette containing SSC as a blank.

3. Place the cuvettes into a waterbath at 25 ° C and allow to temperature equilibrate. Remove the blank, wipe the outside dry and rapidly blank the instrument at 260 nm. Transfer the sample to the

spectrophotometer (be sure to dry and work rapidly) and read the absorbance.

4. Raise the temperature of the bath to 50° C and repeat step .

5. Raise the temperature sequentially to 60° C, 65° C, 70° C, 75° C and 80° C and repeat the absorbance measurements

6. Slowly raise the temperature above 80° and make absorbance measurements every 2° until the absorbance begins to increase. At that point, increase the temperature, but continue to take readings at

1° C intervals

7. Correct all of the absorbance readings for solvent expansion relative to 25° C. 

9. Plot the value of A /A vs temperature and calculate the midpoint of any increased absorbance. This midpoint is the melting point (Tm) for your DNA sample.

Results

Wavelength (nm) Absorbance

220230240250260270280290300

Calculate the GC content of your sample using the formula Percent of G + C = k(Tm -69.3) x 2.44.

Discussion

Questions

1.How pure is your DNA samples? Reflect on the possible sources of contaminations in your DNA samples from different sources?

2. Compare the different melting temperature of DNA obtained from different sources?

3. What does the ratio of AT/CG tells you?

4. Why does the melting temperature of the DNA sample depends on the GC content?