Question

3) You have four tubes containing the same DNA at the sameconcentrationin each tube (pH 7.5) but under different environmental conditions (listed below).  The T_Mfor this DNA molecule is 78°C at physiological salt conditions (~100 mM NaCl):

1. DNA in an aqueous solution that has been digested completely to nucleotideswith DNAse I enzyme.

2. DNA in an aqueous solution containing no salt and has been heated to 78°C.

3. DNA in an aqueous solution containing physiological concentrations of salt and heated to 78°C.

4. DNA in an aqueous solution containing physiological concentrations of salt and is heated only to 37^oC.

Match the samples above with their corresponding OD₂₆₀readings.  Next, explain your reasoning for each of the matches you made.  (For example, “The DNA in sample (a) gives the highest OD reading because…”).

• OD₂₆₀= 1.01 à

• OD₂₆₀= 0.195 à

• OD₂₆₀= 0.713 à

• OD₂₆₀= 0.421 à

Answer 1

3. The melting temperature (Tm) is temperature at which the double stranded DNA will unwind and form a single stranded molecule. When DNA is heated at or above its melting temperature under physiological conditions, the DNA is denatured, forming a single stranded molecule. In this denatured form, the nitrogenous bases will become unstacked. Hence, their absorbance will increase. This is known as hyperchromatic effect.

a. When DNA is completely digested with DNAse I in aqueous solution, the OD260 nm is= 1.01

The DNA in this sample gives highest absorbance.

When DNA is digested with DNase I, the DNase I will cleave the phosphodiester linkage between the nucleotides. This results in formation of deoxyribonucleotides. Deoxyribonucleotides have the highest absorbance as compared to ssDNA or dsDNA. Hence, the highest OD at 260 nm will be obtained when DNase I treatment is performed on dsDNA.

b. The OD260 nm= 0.713 when DNA is heated at 780C in aqueous solution containing no salt.

The DNA in this sample gives the second highest OD at 260 nm because salt concentration affects denaturation. When the salt concentration is lower, DNA will denature faster than when the salt concentration is higher. DNA is a polyanionic molecule and the negative charges on each phosphate are shielded by salt ions. When the salt concentration decreases, there is no shielding effect. Hence, the DNA will denature. At 780C, the melting temperature, DNA is denatured from double stranded to single stranded form. Single stranded DNA absorbs more UV light as the bases are unstacked.

c. The OD260 nm= 0.421 when DNA is heated at 780C under physiological concentrations of salt.

The DNA in this sample has lesser OD at 260 nm than the sample heated at melting temperature with no salt. The salt in the sample will shield the negative charges on each phosphate. Thus, the denaturation is slower. Higher salt concentration favors renaturation. The DNA is in single stranded form and hence, will still show significant absorbance. Single stranded DNA has unstacked nitrogenous bases, thereby absorbing more UV light.

d. the OD 260 nm= 0.195 when DNA is heated in aqueous solution at 370C in physiological salt concentration.

The DNA in this sample gives the least OD because the DNA is not denatured. 370C is quite less than the melting temperature. Hence, no denaturation occurs and the DNA remains in double stranded form. In double stranded DNA, the bases have stacked interactions between them. Thus, ds DNA absorbs the least amount of UV light.