MOLECULAR DIAGNOSTICS EXAM 2
MATERIAL QUESTIONS AND
ANSWERS
If DNA is not pure, then it is usually contaminated with _____________ or
________________ from the DNA isolation reagents. - ANSWER-protein.
inorganic/organic compounds.
Protein absorbs maximally at _____________ nm.
Contaminants (EDTA, phenol, Guanidine salts) absorb maximally at ____________
nm. - ANSWER-A280
A230.
To determine DNA purity from protein contamination, you can take the A260/A280 to
get a ratio. It should be greater than ___________ to use in downstream reactions
(PCR).
To determine DNA purity from inorganic/organic compound contamination, you can
take the A260/A230 to get a ratio. It should be greater than ___________ to use in
downstream reactions (PCR). - ANSWER-1.6
1.8
It is possible to detect proteins and quantitate the concentration of a purified protein
in solution if you know the exact amino acid sequence. - ANSWER-True. Proteins
absorb UV light because of specific residues Trp (W), Phe (F), Tyr (Y). Normally the
absorbance at 280 nm is measured (W, Y, and C residues).
The absorption of UV radiation corresponds to the excitation of _______________.
Transitions involving electrons to a (higher/lower) energy can be used to quantify
organic compounds. - ANSWER-outer electrons.
higher.
______________ is a small UV spectrophotometer that uses 2 uL of nucleic acid to
determination the concentration. It is more sensitive than standard spectrometers in
that no dilutions are made.
It determines the purity using the ratio of what absorbances? - ANSWER-The
Nanodrop
260/280 and 260/230.
What are some disadvantages of using the nanodrop in the clinical lab? - ANSWER-
Tells you nothing about the integrity of the DNA or RNA (i.e. whether it is degraded).
,Readings are influenced by contamination (not specific).
What are some advantages of using the nanodrop in the clinical lab? - ANSWER-
Quick and cheap to run ($10,000) for the instrument, sensitive, moderately accurate,
no reagents needed.
Fluorescence spectroscopy theory:
Some molecules can be excited electronically by a photon of light and move to an
'activated state' S1'. The activated state lasts for a ____________ period of time
during which the molecule loses energy S1. When the molecule comes back down to
the ground state (S0) it emits the energy in the form of light. As a general rule, the
emitted light has a _____________ wavelength than the wavelength that excited the
molecule. Fluorescence is generally much more sensitive than chromagenic
molecules as a single molecule can be excited multiple times. - ANSWER-short.
longer.
What are the 3 (simplified) steps of fluorescence spectroscopy? - ANSWER-1.
absorption.
2. excited or activated state.
3. emission.
In fluorescence spectroscopy, the blue curve = _____________ and the red curve =
_______________. - ANSWER-Hoechst excitation.
Emission spectra.
What dye is used to bind DNA in fluorescence spectroscopy? What is its absorbance
maxiumum? What is its emission maximum? - ANSWER-Hoechst.
Absorbance: 350 nm (UV).
Emission: 461 nm (blue light).
When does Hoechst dye fluoresce? - ANSWER-When intercalated into the minor
groove (wedged into the minor grooves of DNA).
What are the steps to quantify DNA with a fluorometer? - ANSWER-1. Blank
spectrophotometer with working dilution of H33342 or H33258.
2. To the working dilution add a known quantity of DNA. Hit 'Std'.
3. To the working H33342 dilution add your DNA of unknown concentration. Hit
'measure'.
4. Spits out a number.
Which has greater sensitivity- fluorescence spectroscopy or UV spectroscopy? -
ANSWER-Fluorescence has greater sensitivity and much lower concentrations can
be read.
What dyes are used to bind RNA with a fluorometer? - ANSWER-PicoGreen or
RiboGreen fluorescent dyes.
What are the advantages of fluorometry? - ANSWER-Very quantitative and sensitive
(10 pg/uL).
, Specific - Not influenced by protein contamination & can measure DNA or RNA.
What are the disadvantages of fluorometry? - ANSWER-Requires reagents
Dyes are instable in light
Tells you nothing about the integrity of the sample
Gel electrophoresis is the movement of a charged molecule in a matrix by an electric
field. The general principles are:
1. A negatively charged molecule will migrate towards the ______________ and a
positively charged molecule moves toward the _______________.
2. The migration rate of molecules exposed to a current depends on the size of the
molecule, the charge of the molecule and to a lesser extent the shape of a molecule.
- ANSWER-anode (+ charge).
cathode (- charge).
______________ is a polysaccharide purified from seaweed. - ANSWER-Agarose.
--Highly purified agarose is used as a molecular sieve. No charges on it. Smaller
molecule can migrate easier/faster through it than a larger molecule.
Describe the principle of gel electrophoresis. - ANSWER-Mixtures move through a
gel which is porous. The electric field drives the migration.
If all molecules have a similar charge and shape, then the smaller molecules will
migrate faster than larger molecules due to a lower frictional coefficient.
In gel electrophoresis, DNA is negatively charged (____________ backbone). When
placed in an electric field nucleic acids will migrate to the ____________ pole
(anode). - ANSWER-phosphodiester.
positive.
What can gel electrophoresis be used for? - ANSWER-Determine if a nucleic acid is
present in a sample (must use fluorescent compounds).
Quantify the abundance, need a standard.
Examine the integrity, can measure the size/length.
Do fluorescence spectroscopy and gel electrophoresis use the same dyes? -
ANSWER-No.
In Agarose Gel Electrophoresis, the movement of molecules is impeded in the gel so
that molecules will collect or form a band according to their ______________. -
ANSWER-speed of migration.
The concentration of gel/buffer in gel electrophoresis will affect the resolution of
fragments of different size ranges. - ANSWER-True.
--smaller strands migrate faster.
--use a lower % gel when separating larger nucleic acids.
MATERIAL QUESTIONS AND
ANSWERS
If DNA is not pure, then it is usually contaminated with _____________ or
________________ from the DNA isolation reagents. - ANSWER-protein.
inorganic/organic compounds.
Protein absorbs maximally at _____________ nm.
Contaminants (EDTA, phenol, Guanidine salts) absorb maximally at ____________
nm. - ANSWER-A280
A230.
To determine DNA purity from protein contamination, you can take the A260/A280 to
get a ratio. It should be greater than ___________ to use in downstream reactions
(PCR).
To determine DNA purity from inorganic/organic compound contamination, you can
take the A260/A230 to get a ratio. It should be greater than ___________ to use in
downstream reactions (PCR). - ANSWER-1.6
1.8
It is possible to detect proteins and quantitate the concentration of a purified protein
in solution if you know the exact amino acid sequence. - ANSWER-True. Proteins
absorb UV light because of specific residues Trp (W), Phe (F), Tyr (Y). Normally the
absorbance at 280 nm is measured (W, Y, and C residues).
The absorption of UV radiation corresponds to the excitation of _______________.
Transitions involving electrons to a (higher/lower) energy can be used to quantify
organic compounds. - ANSWER-outer electrons.
higher.
______________ is a small UV spectrophotometer that uses 2 uL of nucleic acid to
determination the concentration. It is more sensitive than standard spectrometers in
that no dilutions are made.
It determines the purity using the ratio of what absorbances? - ANSWER-The
Nanodrop
260/280 and 260/230.
What are some disadvantages of using the nanodrop in the clinical lab? - ANSWER-
Tells you nothing about the integrity of the DNA or RNA (i.e. whether it is degraded).
,Readings are influenced by contamination (not specific).
What are some advantages of using the nanodrop in the clinical lab? - ANSWER-
Quick and cheap to run ($10,000) for the instrument, sensitive, moderately accurate,
no reagents needed.
Fluorescence spectroscopy theory:
Some molecules can be excited electronically by a photon of light and move to an
'activated state' S1'. The activated state lasts for a ____________ period of time
during which the molecule loses energy S1. When the molecule comes back down to
the ground state (S0) it emits the energy in the form of light. As a general rule, the
emitted light has a _____________ wavelength than the wavelength that excited the
molecule. Fluorescence is generally much more sensitive than chromagenic
molecules as a single molecule can be excited multiple times. - ANSWER-short.
longer.
What are the 3 (simplified) steps of fluorescence spectroscopy? - ANSWER-1.
absorption.
2. excited or activated state.
3. emission.
In fluorescence spectroscopy, the blue curve = _____________ and the red curve =
_______________. - ANSWER-Hoechst excitation.
Emission spectra.
What dye is used to bind DNA in fluorescence spectroscopy? What is its absorbance
maxiumum? What is its emission maximum? - ANSWER-Hoechst.
Absorbance: 350 nm (UV).
Emission: 461 nm (blue light).
When does Hoechst dye fluoresce? - ANSWER-When intercalated into the minor
groove (wedged into the minor grooves of DNA).
What are the steps to quantify DNA with a fluorometer? - ANSWER-1. Blank
spectrophotometer with working dilution of H33342 or H33258.
2. To the working dilution add a known quantity of DNA. Hit 'Std'.
3. To the working H33342 dilution add your DNA of unknown concentration. Hit
'measure'.
4. Spits out a number.
Which has greater sensitivity- fluorescence spectroscopy or UV spectroscopy? -
ANSWER-Fluorescence has greater sensitivity and much lower concentrations can
be read.
What dyes are used to bind RNA with a fluorometer? - ANSWER-PicoGreen or
RiboGreen fluorescent dyes.
What are the advantages of fluorometry? - ANSWER-Very quantitative and sensitive
(10 pg/uL).
, Specific - Not influenced by protein contamination & can measure DNA or RNA.
What are the disadvantages of fluorometry? - ANSWER-Requires reagents
Dyes are instable in light
Tells you nothing about the integrity of the sample
Gel electrophoresis is the movement of a charged molecule in a matrix by an electric
field. The general principles are:
1. A negatively charged molecule will migrate towards the ______________ and a
positively charged molecule moves toward the _______________.
2. The migration rate of molecules exposed to a current depends on the size of the
molecule, the charge of the molecule and to a lesser extent the shape of a molecule.
- ANSWER-anode (+ charge).
cathode (- charge).
______________ is a polysaccharide purified from seaweed. - ANSWER-Agarose.
--Highly purified agarose is used as a molecular sieve. No charges on it. Smaller
molecule can migrate easier/faster through it than a larger molecule.
Describe the principle of gel electrophoresis. - ANSWER-Mixtures move through a
gel which is porous. The electric field drives the migration.
If all molecules have a similar charge and shape, then the smaller molecules will
migrate faster than larger molecules due to a lower frictional coefficient.
In gel electrophoresis, DNA is negatively charged (____________ backbone). When
placed in an electric field nucleic acids will migrate to the ____________ pole
(anode). - ANSWER-phosphodiester.
positive.
What can gel electrophoresis be used for? - ANSWER-Determine if a nucleic acid is
present in a sample (must use fluorescent compounds).
Quantify the abundance, need a standard.
Examine the integrity, can measure the size/length.
Do fluorescence spectroscopy and gel electrophoresis use the same dyes? -
ANSWER-No.
In Agarose Gel Electrophoresis, the movement of molecules is impeded in the gel so
that molecules will collect or form a band according to their ______________. -
ANSWER-speed of migration.
The concentration of gel/buffer in gel electrophoresis will affect the resolution of
fragments of different size ranges. - ANSWER-True.
--smaller strands migrate faster.
--use a lower % gel when separating larger nucleic acids.
