Section 3 Flashcards
What is the difference in structure between purines and pyrimidines? How might this affect the properties of the nucleic acids that they make up?
Purines have two rings, pyrimidines have one. These rings have alternating single and double bonds, showing resonance.
The resonance imparts partial double-bond character to most of the bonds within the ring structures. As a result, pyrimidines have a planar structure, while purines have a nearly planar structure with a slight curve.
Because of this resonance, the electrons in the rings can absorb UV light at around 260 nm. This is how we detect and measure the concentration of nucleic acids.
What does the molar extinction coefficient (ε) measure, and what are its units?
The molar extinction coefficient (ε) measures the amount of light absorbed by a 1M solution with a light path length of 1 cm. Its units can be expressed as per molar per centimeter (M⁻¹ · cm⁻¹), liters per Molar per centimeter (L · mol⁻¹ · cm⁻¹), or litres per gram per centimeter (g⁻¹ · cm⁻¹ · L). This property is specific to the molecule being measured.
What does the ability of bases to absorb UV light depend on?
It varies depending on whether or not they exist in their free form or part of a nucleic acid
The ability of nitrogenous bases to absorb UV (ultraviolet) light is influenced by their molecular environment. When nitrogenous bases are in their free form, not bound within a nucleic acid, they are more capable of absorbing UV light. This is because, in a free state, each individual base has the freedom to interact with incoming UV photons and absorb the energy.
On the other hand, when these bases are incorporated into a nucleic acid (such as DNA or RNA), they are arranged in a stacked and close-packed manner. This close interaction between adjacent bases within the nucleic acid structure alters their ability to absorb UV light. In this context, the stacked bases collectively absorb less UV light compared to the same concentration of free nucleotides.
What does Beer’s Law state about the relationship between absorbance and concentration in a solution?
Beer’s Law states that the absorbance of light at a certain wavelength is directly proportional to the concentration of the solution. In other words, the darker a solution (higher absorbance), the more concentrated it is as it absorbs more light.
The equation for Beer’s Law is A260 = ε260 * c * l.
What do each of the variables represent?
A260 is the absorbance of UV light (optical density) at 260 nm. It has no units.
ε260 is the extinction coefficient [g⁻¹ · cm⁻¹ · L]. This is the light absorbed by a 1 M solution over a 1 cm path-length.
c represents the concentration of the solution. (mol/L)
l is the path length, or the distance that light travels through a sample in an analytical cell. (cm)
The absorbance value for a nucleic acid solution, as measured by a spectrophotometer, is 1. The
path length is a standard 1 cm, and the extinction coefficient is 0.020 g⁻¹ · cm⁻¹ · L.
What is the concentration of the nucleic acid solution?
ANSWER: 50 g/L
A₂₆₀ = ε₂₆₀ * c * l
c = A₂₆₀ / (ε₂₆₀ * l)
Given:
ε₂₆₀ = 0.020 g⁻¹·cm⁻¹·L
A₂₆₀ = 1 (as per your calculations)
l = 1 cm
Now, plug in the values:
c = 1 / (0.020 g⁻¹·cm⁻¹·L * 1 cm)
c = 1 / 0.020 g⁻¹·L
c = 50 g/L
So, the concentration (c) of the solution is 50 grams per liter (g/L).
How does the absorption of UV light differ between single-stranded (ss) and double-stranded (ds) DNA, and how does it compare to free nucleotides?
The absorption of UV light varies depending on whether the DNA is single-stranded (ss) or double-stranded (ds).
The order of the amount of UV absorption is as follows: dsDNA < ssDNA «_space;free nucleotides.
What does the hyperchromic effect in DNA refer to, and when does it occur?
The hyperchromic effect refers to the large increase in light absorption at 260 nm that occurs as double-helical DNA unwinds (or melts). This happens when DNA denatures, and the base pairs are disrupted, leading to the separation of the two strands, forming single-stranded DNA (ssDNA).
Why does single-stranded DNA (ssDNA) exhibit higher UV light absorption than double-stranded DNA (dsDNA) at the same concentration during the hyperchromic effect?
During the hyperchromic effect, when DNA transitions from dsDNA to ssDNA, the resonance within the bases in each strand is no longer constrained. As a result, the UV light absorption of ssDNA becomes higher than that of dsDNA at the same concentration.
How can the transition of DNA from double-stranded to single-stranded (or vice versa) be detected?
The transition of DNA from double-stranded to single-stranded (or vice versa) can be detected by monitoring the change in absorption of UV light, specifically at 260 nm. The hyperchromic effect, with increased UV absorption during denaturation, is a key indicator of this transition.
What is the hypochromic effect in DNA, and when does it occur?
The hypochromic effect refers to the significant decrease in light absorption at 260 nm that occurs as single strands of DNA anneal to form double-helical DNA. This effect is observed when the forces stabilizing the DNA double helix, such as hydrogen bonding and base stacking, limit the amount of resonance that can occur within the aromatic rings of the bases. As a result, there is a reduced amount of UV light absorbed as single-stranded DNA (ssDNA) anneals to form double-stranded DNA (dsDNA).
Identify the correct relative order of UV absorption (from most to least)
Response recorded
Free dNTPs > dsDNA > ssDNA
Free dNTPs > ssDNA > dsDNA
ssDNA > dsDNA > Free dNTPs
dsDNA > ssDNA > Free dNTPs
None of the above
Free dNTPs > ssDNA > dsDNA
most absorbance occurs with the most electron freedom, which means least constraint
What is the melting temperature (Tm) of DNA, and how is it defined?
The melting temperature (Tm) is the temperature at which 50% of the DNA is in the single-stranded form (ssDNA), and the other 50% is in the double-stranded form (dsDNA). It’s a critical parameter in DNA analysis and can be calculated from the absorbance data, often measured at 260 nm.
What happens to the ability of DNA to resonate as it transitions from a double-stranded to a single-stranded state, and how does this change affect its interaction with light?
As DNA shifts from a double-stranded to a single-stranded configuration, it gains a greater capacity to resonate. This heightened resonance and enhanced freedom of electrons lead to a greater absorption of light.
Does DNA absorb more UV light in its single stranded for or double stranded form?
DNA absorbs more light in its single stranded form, as the resonance within the nitrogenous bases are no longer constrained by the forces that stabilize the DNA double helix.
