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BHCM218 > Module 03 - Section 03 > Flashcards

Module 03 - Section 03 Flashcards

Quantification of Nucleic Acids

1
Q

Why is quantification of nucleic acids performed?

A

Determine the concentration of DNA and RNA in a sample

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2
Q

How are purines and pyrimidines able to absorb UV light?

A

As a result of resonance, the delocalized electrons within the conjugated ring systems are available to absorb UV light at wavelengths near 260 nm

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3
Q

Are purines and pyrimidines tetrahedral or planar?

A

Purines: nearly planar
pyrimidines: planar

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4
Q

Describe spectophotometry

A

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

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5
Q

What is the molar extinction coefficient?

A

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

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6
Q

Which NMP absorbs the most light at 260nm?

A

Adenosine MonoPhosphate (AMP)

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7
Q

Which NMP absorbs the least amount of light at 260nm?

A

Cytosine monophosphate (CMP)

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8
Q

What are the molar coefficient of AMP, GMP, UMP, dTMP, CMP at 260nm?

A 
AMP: 15400
GMP: 11700
UMP: 9900
dTMP: 9200
CMP: 7500
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9
Q

Do nitrogenous bases absorb more UV light when in free form or bound within a nucleic acid and why?

A

Free form, because the close interactions of stacked bases when bound decreases the absorption of UV light

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10
Q

What is Beer’s law?

A

The absorbance of light at a certain wavelength is directly proportional to the concentration of the solution - more concentrated = more light absorbed

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11
Q

What is the equation of Beer’s law?

A

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

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12
Q

What is the optical density?

A

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

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13
Q

What is the relative order of UV absorption between double stranded DNA, single stranded DNA and free nucleotides

A

dsDNA < ssDNA&laquo_space;free nucleotides

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14
Q

What is hypochromicity?

A

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

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15
Q

What is hyperchromicity?

A

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

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16
Q

Why can transition from dsDNA to ssDNA (or vice versa) be detected?

A

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

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17
Q

What is denaturation?

A

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

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18
Q

Describe the 2 steps of change when DNA is exposed to extreme pH or temperature above 80 degree celsius

A

(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

19
Q

Describe dsDNA at pH= and temperature=25 degrees

A

highly viscous

20
Q

How can you determine the melting point (Tm) - or the temperature at which half the DNA in the sample is denatured

A

Tracking the absorbance of an aqueous solution across a range of temperatures

21
Q

How does base composition affect the melting point?

A

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

22
Q

Which regions of the dsDNA will denature first?

A

A=T rich regions

23
Q

List 2 reasons why we would want to denature DNA in the lab

A

(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

24
Q

What is the equation to determine melting point?

A

Tm = 0.41 (%G+C) + 69.3 celsius

25
Q

What is renaturation?

A

Reconstruction of a nucleic acid (or protein) to its original form - particularly after denaturation

26
Q

Describe the process of renaturation when strands are not completely separated

A

Rapid, 1-step process, as long as the 2 strands are not completely separated and at least a dozen residues must remain intact in order to unite the 2 strands
When pH/Temp returns to normal, the separated segments spontaneously rewind to form an intact duplex (aka reannealing)

27
Q

Describe the process of renaturation when strands are completely separated

A

(1) Relatively slow step: complementary sequences in the two strands must “find” each other by random collision and for a short segment of double helix – if they dont find each other properly a distorted dsDNA molecule may result
(2) Fast step: remaining unpaired bases successively come into register as base pairs, and the 2 strands zipper themselves together

28
Q

What are Duplexes and RNA-DNA hybrids?

A

nucleic acids from different species of animals can form hybrid duplexes

29
Q

Describe the reaction of hybridization

A

2 samples of isolated DNA or RNA are heated to cause denaturation and then mixed. The sample is left to cool, allowing complementary strands to form a duplex whether it is a DNA-RNA or DNA-DNA or RNA-RNA hybrid

30
Q

Which kind of duplexes is more stable?

A

RNA duplexes are more stable than DNA duplexes due to the extra 2’OH that provides another point for hydrogen bonding, then RNA-DNA duplexes and then DNA-DNA

31
Q

How does salt concentration affect the melting point of DNA

A

Salt stabilizes the duplex by stabilizing the negative charge on the backbone –
Raises the melting point temperature in a linear fashion proportionally to the salt concentration

32
Q

How do organic solvents affect he melting point of DNA?

A

Increasing organic solvents in a solution lower the melting temperature by destabilizing the duplex

33
Q

How does DNA concentration affect the melting point ?

A

DNA concentration affects the absorbance value but not the melting temperature, since this is a property of the GC content

34
Q

What are the 3 factors that influence DNA hybridization?

A

DNA concentration, pH, and base pair mismatch between the 2 strands

35
Q

How does DNA concentration affect hybridization?

A

More concentrated sample of DNA has more frequent intermolecular collisions, and therefore, a faster hybridization rate

36
Q

How does base pair mismatch affect hybridization?

A

When hybridizing 2 nucleic acid segments, that are completely complementary, mismatched nucleotides can interrupt base pairing, stacking interaction and DNA stability

37
Q

How does pH affect hybridization?

A

In 5

38
Q

What is the stringency of DNA hybridization?

A

Extent that hybridization can occur between two non-complementary strands of nucleic acids
–> degree of complementarity required between two strand in order for them to hybridize

39
Q

What is high stringency?

A

Hybridization occurs only when the two strands are highly compatible

40
Q

What is low stringency?

A

Hybridization occurs even in the presence of some base mismatches

41
Q

How can stringency of DNA hybridization be altered?

A

Using the same factors that altered dsDNA duplex stability: temperature, salt, and organic solvent

42
Q

What factors increase stringency?

A

Duplex formation not favoured:

  • High temperatures (close to Tm of hybrid)
  • Low-salt concentrations
  • Presence of organic solvents
43
Q

What factors decrease stringency?

A

Hybrid formation favoured:

  • Low temperature (below Tm of the duplex)
  • High-salt concentrations
  • Absence of organic solvents

BHCM218 (24 decks)

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