MOLECULAR DIAGNOSTICS EXAM 2
MATERIAL QUESTIONS WITH
CORRECT ANSWERS
Given the percent agar, determine the DNA size resolved (in base pairs):
1. 0.8%
2. 1.0%
3. 1.2 %
4. 2.0% - ANSWER-1. 500-3000
2. 350-2000
3. 225-1000
4. 75-500
What is 0.8% agar used for?
What is 2.0% agar used for? - ANSWER-Assessing genomic DNA integrity, large
DNA fragments.
Small PCR products.
Name 2 buffers used in agarose gel electrophoresis. - ANSWER-TAE (Tris-acetate
EDTA).
TBE (Tris-borate EDTA).
Which buffer in gel electrophoresis is effective for running larger sized DNA
fragments? Which buffer resolves small fragments (<200 bp). - ANSWER-TAE is
larger.
TBE is smaller.
How do the TAE and TBE buffers work in gel electrophoresis? - ANSWER-Both
buffers bind divalent catio.ns such that DNA has a uniform negative charge and runs
strictly according to size
In agarose gel electrophoresis, the rate of migration of a DNA fragment in an electric
field depends on _______________. - ANSWER---Voltage (75-150 Volts)
--Agar %
--Buffer used (tris acetate runs faster than tris borate).
--Size of DNA in base pairs (smaller fragment = runs faster).
What are the steps for running gel electrophoresis? - ANSWER-1. Prepare correct
percent agarose gel (stain). Place fluorescent stain (dye) in gel (ethidium
bromide/SYBR gold).
2. Place in chamber and add buffer (TAE or TBE).
,3. Mix DNA with loading dye which contains... 30% sucrose, TE buffer (10 mM Tris
pH8.0, 1mM EDTA), color indicator (bromophenol blue, xylene cyanol, cresol red or
any other food dye).
4. Load DNA into well.
5. Start the current flowing
6. Visualize (or stain, then visualize)
What dye is used in agarose gel electrophoresis? - ANSWER-ethidium bromide or
SYBR gold.
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.
Name 3 methods for DNA quantification. - ANSWER-UV spectroscopy
Fluorometry
Gel electrophoresis
The goal of ______________ is to measure the light of a specific wavelength
absorbed by an analyte (chromophore). - ANSWER-absorbance spectroscopy.
The light emitted from an analyte (fluorophore) following the excitation by a light of a
specific wavelength is measured by _______________. - ANSWER-fluorescence
spectroscopy.
A ____________________ is a tool that can be used to measure both absorbance
and fluorescence. - ANSWER-spectrophotometer.
Absorption is due to interaction of light with electronic and vibrational modes of the
molecule. Light is a form of electromagnetic radiation.
, Absorbance spectroscopy uses ___________ and ____________ radiation. -
ANSWER-ultraviolet and visible.
UV wavelengths are ____________ nm in length.
Visible wavelengths are from __________ to ___________ nm. - ANSWER-180 to
350.
350 (violet) to 700 nm (red).
How is absorption spectroscopy used to quantify DNA? - ANSWER-UV quantification
of nucleic acid concentration, detects nucleic acids and quantifies its concentration of
DNA in solution.
2-5 ng/uL detection limit.
In absorption spectroscopy, DNA absorbs UV light maximally at ___________ nm. -
ANSWER-260 nm.
Given the nucleic acid, determine the absorbance reading at 260 nm:
1. double stranded DNA
2. RNA
3. single stranded DNA - ANSWER-1/extinction coefficient (ng/uL)
1. 50 ng/uL
2. 40 ng/uL
3. 33 ng/uL
DNA has just been isolated from paraffin-embedded tissue block (100 uL final
volume). 10 uL of DNA was used in a 500 uL final volume to obtain an absorbance at
260 nm of 0.022 in a standard spectrophotometer (1 cm path length) with a quartz
cuvette.
A. What is the DNA concentration?
B. What is the DNA yield? - ANSWER-A. Absorbance at 260 X [1/extinction
coefficient] X dilution factor = [DNA] ng/uL. --> 0.022 x 50 ng/uL x (500/10) = 55
ng/uL
B. A.Yield (how much DNA was isolated). 55 ng/uL x 100 uL = 5500 ng or 5.5 ug
What is the formula to calculate DNA concentration? - ANSWER-Absorbance at 260
X [1/extinction coefficient] X dilution factor = [DNA] ng/uL.
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.
MATERIAL QUESTIONS WITH
CORRECT ANSWERS
Given the percent agar, determine the DNA size resolved (in base pairs):
1. 0.8%
2. 1.0%
3. 1.2 %
4. 2.0% - ANSWER-1. 500-3000
2. 350-2000
3. 225-1000
4. 75-500
What is 0.8% agar used for?
What is 2.0% agar used for? - ANSWER-Assessing genomic DNA integrity, large
DNA fragments.
Small PCR products.
Name 2 buffers used in agarose gel electrophoresis. - ANSWER-TAE (Tris-acetate
EDTA).
TBE (Tris-borate EDTA).
Which buffer in gel electrophoresis is effective for running larger sized DNA
fragments? Which buffer resolves small fragments (<200 bp). - ANSWER-TAE is
larger.
TBE is smaller.
How do the TAE and TBE buffers work in gel electrophoresis? - ANSWER-Both
buffers bind divalent catio.ns such that DNA has a uniform negative charge and runs
strictly according to size
In agarose gel electrophoresis, the rate of migration of a DNA fragment in an electric
field depends on _______________. - ANSWER---Voltage (75-150 Volts)
--Agar %
--Buffer used (tris acetate runs faster than tris borate).
--Size of DNA in base pairs (smaller fragment = runs faster).
What are the steps for running gel electrophoresis? - ANSWER-1. Prepare correct
percent agarose gel (stain). Place fluorescent stain (dye) in gel (ethidium
bromide/SYBR gold).
2. Place in chamber and add buffer (TAE or TBE).
,3. Mix DNA with loading dye which contains... 30% sucrose, TE buffer (10 mM Tris
pH8.0, 1mM EDTA), color indicator (bromophenol blue, xylene cyanol, cresol red or
any other food dye).
4. Load DNA into well.
5. Start the current flowing
6. Visualize (or stain, then visualize)
What dye is used in agarose gel electrophoresis? - ANSWER-ethidium bromide or
SYBR gold.
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.
Name 3 methods for DNA quantification. - ANSWER-UV spectroscopy
Fluorometry
Gel electrophoresis
The goal of ______________ is to measure the light of a specific wavelength
absorbed by an analyte (chromophore). - ANSWER-absorbance spectroscopy.
The light emitted from an analyte (fluorophore) following the excitation by a light of a
specific wavelength is measured by _______________. - ANSWER-fluorescence
spectroscopy.
A ____________________ is a tool that can be used to measure both absorbance
and fluorescence. - ANSWER-spectrophotometer.
Absorption is due to interaction of light with electronic and vibrational modes of the
molecule. Light is a form of electromagnetic radiation.
, Absorbance spectroscopy uses ___________ and ____________ radiation. -
ANSWER-ultraviolet and visible.
UV wavelengths are ____________ nm in length.
Visible wavelengths are from __________ to ___________ nm. - ANSWER-180 to
350.
350 (violet) to 700 nm (red).
How is absorption spectroscopy used to quantify DNA? - ANSWER-UV quantification
of nucleic acid concentration, detects nucleic acids and quantifies its concentration of
DNA in solution.
2-5 ng/uL detection limit.
In absorption spectroscopy, DNA absorbs UV light maximally at ___________ nm. -
ANSWER-260 nm.
Given the nucleic acid, determine the absorbance reading at 260 nm:
1. double stranded DNA
2. RNA
3. single stranded DNA - ANSWER-1/extinction coefficient (ng/uL)
1. 50 ng/uL
2. 40 ng/uL
3. 33 ng/uL
DNA has just been isolated from paraffin-embedded tissue block (100 uL final
volume). 10 uL of DNA was used in a 500 uL final volume to obtain an absorbance at
260 nm of 0.022 in a standard spectrophotometer (1 cm path length) with a quartz
cuvette.
A. What is the DNA concentration?
B. What is the DNA yield? - ANSWER-A. Absorbance at 260 X [1/extinction
coefficient] X dilution factor = [DNA] ng/uL. --> 0.022 x 50 ng/uL x (500/10) = 55
ng/uL
B. A.Yield (how much DNA was isolated). 55 ng/uL x 100 uL = 5500 ng or 5.5 ug
What is the formula to calculate DNA concentration? - ANSWER-Absorbance at 260
X [1/extinction coefficient] X dilution factor = [DNA] ng/uL.
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.
