The Effect of Metal Ions of Varying Sizes and Concentrations on DNA Melting Temperature

George Schank

Abstract

As DNA is heated in solution, it begins to denature.  The native double helix form of DNA dissociates into single strands (melting).  Many factors affect the temperature at which DNA transforms from a double stranded form to a single stranded form.  The presence of cations is one such factor that can have a stabilizing affect on the double helix form of DNA. 

The size, charge, and concentration of the cation in solution are key factors that effect the stabilization of DNA.  This experiment tested each of these variables with respect to ions having different size and charge (K⁺, Mg²⁺, Ca²⁺, Ba²⁺, and Zn²⁺) in order to determine the ion with the most effective combination of charge and size that increases the melting temperature.  This was accomplished by altering one variable while the other remained constant.

It was determined that potassium and barium had no effect on the melting temperature, while magnesium and calcium increased the melting temperature.  Furthermore, magnesium outperformed calcium and was found to be the most effective ion at higher concentrations for stabilizing DNA.

Introduction

DNA (deoxyribonucleic acid) is a nucleic acid that carries genetic information and has a double helix structure.  It is made up of a chain of nucleotides which contains a sugar, a phosphate, and a base.  The four variations of nucleotides are: adenine, thymine, guanine, and cytosine.  Adenosine and thymine bind together (with two hydrogen bonds) and guanine and cytosine bind together (with three hydrogen bonds).  This is significant when the stability of the double-helix is examined.  The H-bonding between the bases must be overcome if the DNA is to “melt” from its double-helix form into a single strand.  There are many factors which can influence the overall stability of DNA, such as G-C content, chain length, and environment (temperature, pH, presence of ions, etc.). 

It is common knowledge that the higher the content of guanine and cytosine bonds, the more stable the DNA.  This is due to G-C bonds having three hydrogen bonds instead of only two, like A-T bonds.  It was also suggested that higher cation concentrations will behave in the same manner and stabilize the DNA by forming a shell around the negatively charged phosphates of DNA. This would potentially result in proportionally higher melting temperatures, depending on the concentration (Resendis-Antonio, 2003).  However, the effects of cations of differing sizes and concentrations have not been extensively compared. It is reasonable to expect that the larger the cation, the less extensive the effect it will have on the DNA since larger ions are not expected to cluster around the backbone as extensively as the smaller cations.

Denaturation is a process in which a molecule undergoes a transformation and is no longer in its native state.  This can be caused by heat, acids, bases, detergents, or other specific chemicals.  When heat is the source of denaturation, it is, in the case of DNA, associated with the melting temperature.  DNA’s melting temperature (T_m) can be defined as the temperature at which half of the DNA molecules in a solution have dissociated into single strand molecules.

There have been a number of studies conducted in the past to determine the melting point of DNA.  For instance, it had been previously established by an earlier study by Owczarzy, et al, that the sodium ion stabilizes the backbone structure of DNA (Owczarzy, et al, 2004).  It stabilizes the backbone because the phosphate groups are moderately strong acids, and thus they are almost completely ionized at physiological pH, which allows interactions between the backbone and the cations. Stabilization of the backbone affects the temperature at which the DNA denatures (i.e. the more the ionic interaction, the more stable the DNA and the higher the melting temperature).  Many experiments have been performed involving DNA, its melting temperature, and the factors that affect the T_m.  However, availability of different approaches indicates that there is still much to explore concerning this type of experiment. 

This experiment investigates the effects of potassium, magnesium, calcium, barium, and zinc ions on the stability of the double-helix.  The results of this study potentially may lead to more types of applications in clinical, medicinal, pharmaceutical, and forensic research, as well as in genetic engineering. 

DNA has a maximum absorbance at a wavelength of 260 nm.  Single stranded form has a higher molar absorptivity than the double stranded form at the same wavelength.  In this study, this difference in absorptivities is used to measure the melting temperature by graphing the change in absorbance as the double stranded DNA denatures to single stranded DNA with increasing temperature.

Materials and Methods

Materials:

-Herring sperm DNA from Promega

            -size range: 100-3000bp (for at least 80% of the DNA fragments)

            -concentration: 10 mg/mL (original concentration)

-Calcium chloride, potassium chloride, zinc chloride, barium chloride, and magnesium chloride (all reagent grade)

-Equipment: SP 2000 UV-Vis, Thermo-Spectronic heating sleeve, Thelco water bath (model 85), pump

Procedure:

The absorbance of the standard solution of DNA (0.008 mg/µL) at 260 nm was measured using UV-Vis spectrophotometer at different temperatures ranging from 25^o to 100^o C.

The DNA-metal ion solutions tested contained 0.02 mg of DNA and different concentrations  (10^-4, 10^-3, or 10^-2 M) of metal ions.  Absorbance measurements were done using a blank solution of the respective metal ion concentration at 260 nm, at the temperature range of  25^o to 100^o C.

Experimental Data and Results

The initial concentration of the metal ion ranged from 10^-4 M to 10 ^-2 M.  The concentration of DNA used for this comparison was 0.008 mg/µL due to the known linearity of the standard curve at low concentrations. 

Table 1: Absorbances of DNA solutions without metal ion at 260 nm

Graph 1: Absorbances of DNA solutions without metal ion at 260 nm

Table 2: Absorbances of DNA solution containing K⁺ ions at 260 nm

Graph 2: Absorbances of DNA solution containing K⁺ ions at 260 nm

*Series 1 = 1 x 10^-2 M K⁺; Series 2 = 1 x 10^-3 M K⁺; Series 3 = 1 x 10^-4 M

 Table 3: Absorbances of DNA solution containing Mg²⁺ ions at 260 nm

Graph 3: Absorbances of DNA solution containing Mg²⁺ ions at 260 nm

*Series 1 = 1 x 10^-2 M Mg²⁺; Series 2 = 1 x 10^-3 M Mg ²⁺; Series 3 = 1 x 10^-4

 Table 4: Absorbances of DNA solution containing Ca²⁺ ions at 260 nm

Graph 4: Absorbances of DNA solution containing Ca²⁺ ions at 260 nm

*Series 1 = 1 x 10^-2 M Ca²⁺; Series 2 = 1 x 10^-3 M Ca²⁺; Series 3 = 1 x 10^-4 M Ca²⁺

Table 5: Absorbances of DNA solution containing Ba²⁺ ions at 260 nm