Molecular Biology Tools & Techniques
UPDATED ACTUAL Questions and
CORRECT Answers
GFP - CORRECT ANSWER - Joining the gene for this protein to a protein of interest (by
recombinant DNA) allows for tracking of its location and function in live cells.
FRAP - CORRECT ANSWER - Used to study movement of GFP-labeled proteins. Region
of interest in a cell expressing a GFP-labeled protein is bleached by exposure to high-intensity
light. Fluorescence recovers over time due to the movement of unbleached GFP-labeled
molecules into the bleached region, allowing the rate at which the protein moves within the cell
to be determined.
FRET - CORRECT ANSWER - Used to analyze the interactions of two proteins within a
cell. Two proteins of interest are coupled to different fluorescent dyes, such as two variants of
GFP. GFP variants are chosen to absorb and emit distinct wavelengths of light, such that the light
emitted by one GFP variant excites the second. Interaction between the two can be detected by
illuminating the cell with a wavelength of light that excites the first GFP variant and analyzing
the wavelength of emitted light. If the proteins coupled to these GFP variants interact within the
cell, the fluorescent molecules will be brought close together, and the light emitted by the first
will excited the setting, resulting in light emission at the wavelength characteristic of the second
GFP variant.
Confocal microscopy - CORRECT ANSWER - Allows images of increased contrast and
detail to be obtained by analyzing fluorescence from only a single point in the specimen.
Multi-photon excitation microscopy - CORRECT ANSWER - Alternative to confocal
microscopy that can be applied to living cells. Specimen illuminated with light that causes
simultaneous absorption of two or more photons, which, statistically, will only be significant in
one area.
Electron microscope - CORRECT ANSWER - Can achieve much greater resolution than a
light microscope due to the longer wavelength of life.
, Transmission electron microscopy - CORRECT ANSWER - Specimens fixed and stained
with salts of heavy metals which provide contrast by scattering electrons.
Electron tomography - CORRECT ANSWER - Generates 3D images by computer analysis
of multiple 2D images
Positive vs. negative staining - CORRECT ANSWER - One is used to dye tissue specimen
to determine subcellular location. The other dyes around the speciment in order to visualize
intact structures.
Metal shadowing - CORRECT ANSWER - Specimen coated with thin layer of evaporated
metal, at an angle, so some coated more heavily than others, giving 3D appearance
Freeze fracture - CORRECT ANSWER - Specimen frozen in liquid nitrogen, which splits
the lipid biylayer, revealing interior faces of a cell membrane.
Scanning electron microscopy - CORRECT ANSWER - Provides 3D image of cells by
scanning across specimen with beam of electrons.
X-ray crystallography - CORRECT ANSWER - A high-resolution technique that can
determine the arrangement of individual atoms within a molecule.
Antisense RNA (ssDNA) - CORRECT ANSWER - Blocks translation of mRNA via
transfection of the complementary RNA strand.
RNA interference (RNAi) - CORRECT ANSWER - Uses siRNA to target mRNA for
degradation by use of a RISC. The mRNA is not translated because it is cleaved.
Dominant-inhibitory proteins - CORRECT ANSWER - Block the function of normal
protein by formation of a nonfunctional mutant protein complex.
UPDATED ACTUAL Questions and
CORRECT Answers
GFP - CORRECT ANSWER - Joining the gene for this protein to a protein of interest (by
recombinant DNA) allows for tracking of its location and function in live cells.
FRAP - CORRECT ANSWER - Used to study movement of GFP-labeled proteins. Region
of interest in a cell expressing a GFP-labeled protein is bleached by exposure to high-intensity
light. Fluorescence recovers over time due to the movement of unbleached GFP-labeled
molecules into the bleached region, allowing the rate at which the protein moves within the cell
to be determined.
FRET - CORRECT ANSWER - Used to analyze the interactions of two proteins within a
cell. Two proteins of interest are coupled to different fluorescent dyes, such as two variants of
GFP. GFP variants are chosen to absorb and emit distinct wavelengths of light, such that the light
emitted by one GFP variant excites the second. Interaction between the two can be detected by
illuminating the cell with a wavelength of light that excites the first GFP variant and analyzing
the wavelength of emitted light. If the proteins coupled to these GFP variants interact within the
cell, the fluorescent molecules will be brought close together, and the light emitted by the first
will excited the setting, resulting in light emission at the wavelength characteristic of the second
GFP variant.
Confocal microscopy - CORRECT ANSWER - Allows images of increased contrast and
detail to be obtained by analyzing fluorescence from only a single point in the specimen.
Multi-photon excitation microscopy - CORRECT ANSWER - Alternative to confocal
microscopy that can be applied to living cells. Specimen illuminated with light that causes
simultaneous absorption of two or more photons, which, statistically, will only be significant in
one area.
Electron microscope - CORRECT ANSWER - Can achieve much greater resolution than a
light microscope due to the longer wavelength of life.
, Transmission electron microscopy - CORRECT ANSWER - Specimens fixed and stained
with salts of heavy metals which provide contrast by scattering electrons.
Electron tomography - CORRECT ANSWER - Generates 3D images by computer analysis
of multiple 2D images
Positive vs. negative staining - CORRECT ANSWER - One is used to dye tissue specimen
to determine subcellular location. The other dyes around the speciment in order to visualize
intact structures.
Metal shadowing - CORRECT ANSWER - Specimen coated with thin layer of evaporated
metal, at an angle, so some coated more heavily than others, giving 3D appearance
Freeze fracture - CORRECT ANSWER - Specimen frozen in liquid nitrogen, which splits
the lipid biylayer, revealing interior faces of a cell membrane.
Scanning electron microscopy - CORRECT ANSWER - Provides 3D image of cells by
scanning across specimen with beam of electrons.
X-ray crystallography - CORRECT ANSWER - A high-resolution technique that can
determine the arrangement of individual atoms within a molecule.
Antisense RNA (ssDNA) - CORRECT ANSWER - Blocks translation of mRNA via
transfection of the complementary RNA strand.
RNA interference (RNAi) - CORRECT ANSWER - Uses siRNA to target mRNA for
degradation by use of a RISC. The mRNA is not translated because it is cleaved.
Dominant-inhibitory proteins - CORRECT ANSWER - Block the function of normal
protein by formation of a nonfunctional mutant protein complex.
