Module 03 - Section 03 Flashcards
Quantification of Nucleic Acids (43 cards)
Why is quantification of nucleic acids performed?
Determine the concentration of DNA and RNA in a sample
How are purines and pyrimidines able to absorb UV light?
As a result of resonance, the delocalized electrons within the conjugated ring systems are available to absorb UV light at wavelengths near 260 nm
Are purines and pyrimidines tetrahedral or planar?
Purines: nearly planar
pyrimidines: planar
Describe spectophotometry
Amount of light absrobed by a sample can be measured using a spectrophometer
Sample containing nucleic acids is placed inside a special chamber and exposed to UV light (260nm)
Photo-detector measures how much light passes through the sample and this is represented on an absorption spectrum
Absorption is measure between 230 and 280nm to generate the spectrum
What is the molar extinction coefficient?
Measures the amount og light absorbed by 1M solution with a light path length of 1cm and is a property that is specific to the molecule being measured
Which NMP absorbs the most light at 260nm?
Adenosine MonoPhosphate (AMP)
Which NMP absorbs the least amount of light at 260nm?
Cytosine monophosphate (CMP)
What are the molar coefficient of AMP, GMP, UMP, dTMP, CMP at 260nm?
AMP: 15400 GMP: 11700 UMP: 9900 dTMP: 9200 CMP: 7500
Do nitrogenous bases absorb more UV light when in free form or bound within a nucleic acid and why?
Free form, because the close interactions of stacked bases when bound decreases the absorption of UV light
What is Beer’s law?
The absorbance of light at a certain wavelength is directly proportional to the concentration of the solution - more concentrated = more light absorbed
What is the equation of Beer’s law?
A(260) = e(260) x c x l, where
A= absorbance of UV light as determined by spectrophometer
e(260)=exctintion coefficient in (g^-1 cm^-1 L)
c=concentration
l=path length, or the distance that light travels through a sample in an analytical cell
What is the optical density?
OD, or the amount of UV light able to pass through a solution, at 260nm is a summation of the optical properties of the bases in the molecule
*Not equivalent to absorbance, but will be used interchangeably in this course because. Absorbance of DNA is measure at 260 nm because it has the greatest OD at this wavelength
What is the relative order of UV absorption between double stranded DNA, single stranded DNA and free nucleotides
dsDNA < ssDNA«_space;free nucleotides
What is hypochromicity?
Large decrease in light absorption at 260nm occurring as single strand of DNA anneal to form double-helical DNA
due to forces that stabilize the DNA double helix (hydrogen bonds and base stacking) limit the amount of resonance that can occur within the aromatic rings
= decrease in UV light absorption when ssDNA anneal to form dsDNA
What is hyperchromicity?
Large increase in light absorption at 260nm that occurs as a double-helical DNA unwinds (or melts)
due to the removal of constraints for the resonance in each strand
= more UV light can be absorbed
Why can transition from dsDNA to ssDNA (or vice versa) be detected?
The addition or the removal of constraints by forces that stabilize the double helical structure influences the amount of light that can be absorbed
removal = more light absorbed
addition= less light absorbed
What is denaturation?
The partial of complete unfolding of the specific native conformation of a polypeptide chain, protein or nucleic acids such that the function of the molecule is lost
Describe the 2 steps of change when DNA is exposed to extreme pH or temperature above 80 degree celsius
(1) Denaturation: dsDNA begins to denature and the 2 strands break apart
(2) separation of strands: hydrogen bonds and base stacking interactions are disrupted, causing the double helix to unwind. The result is 2 single strands of DNA
Describe dsDNA at pH= and temperature=25 degrees
highly viscous
How can you determine the melting point (Tm) - or the temperature at which half the DNA in the sample is denatured
Tracking the absorbance of an aqueous solution across a range of temperatures
How does base composition affect the melting point?
GC base pairs have 3 hydrogen bonds between them and AT only 2. Thus, GC base pairs require more energy to break than AT.
the higher the GC content the higher the Tm
Which regions of the dsDNA will denature first?
A=T rich regions
List 2 reasons why we would want to denature DNA in the lab
(1) Analyizing the structure and function of DNA specifically the interactions holding the 2 strands together
(2) Estimating the amount of GC bases, since GC rich DNA will have a higher melting point
What is the equation to determine melting point?
Tm = 0.41 (%G+C) + 69.3 celsius
