BCH 5413 Exam 1
The number of bases per turn of DNA is___________ - correct answer ✔✔~10.5
What does the Meselson and Stahl experiment confirm? - correct answer ✔✔DNA replication is semi-
conservative
Heavy and light strands with nitrogen isotopes prove
True or False
a. Most biological DNA is positively supercoiled.
b. Triplex DNA is pyrimidine rich
c. Chargaff's rule: A=G & T=C
d. A single zinc finger can recognize 10bp of DNA - correct answer ✔✔A. False, negatively = Left-handed.
Some is right handed = positive
B. True
C. False A=T C=G
D. False, 3 bp, use multiple zinc fingers in a row for more sequence specificity/bp
DNA is not a uniform structure, many localized variants
can substitute protein domains, alpha helix/beta hairpin, in zinc fingers to make hybrids with different
binding specificities (designated by alpha helix) for major grooves of DNA
x-ray crystallography allows us to view and design custom designer DNA-binding proteins = alter gene fxn
in intact cells
In A & Z DNA:
a. Which is right handed helix?
,b. Which one has 12bp per helical turn?
c. Which is the dehydrated form? - correct answer ✔✔a. A
b. Z
c. A
primary DNA is B
Gel electrophoresis - correct answer ✔✔molecular separation technique
can separate nucleic acids or proteins
make AGAROSE gel with slots
hot liquid poured with "teeth"
put DNA in slot, run electric current at neutral pH
DNA = negatively charged (phosphates) = migrates towards positive end
key is friction
DNA stained with fluorescent dye, looked at under UV
unknown DNA electrophoresed parallel with known to determine size
distance migrated = can plot on graph with log of molecular weights or number of bp
ex: 20mm = 910bp
PFGE = Pulsed Field Gel Electrophoresis - correct answer ✔✔for longer sequences
,as large as some chromosomes in yeast
What is PAGE? - correct answer ✔✔Polyacrylamide Gel Electrophoresis
used for PROTEINS
have to denature the protein = treat with detergent, SDS, sodium dodecyl sulfate
SDS-PAGE
SDS advantages:
1) coats polypeptides with (-) charge
2) masks natural charge so results are purely based on molecular weight
What is 2-D gel electrophoresis? - correct answer ✔✔LESS POWER: use two different pH values to
compare, pH affects net charge so different rates of mobility
2-D = 1) proteins electrophoresed through narrow tube gel with pH gradients by ampholytes. Once
protein becomes neutral, no longer moves = isoelectric point
2) gel removed from tube, placed at top of regular gel for SDS-PAGE
What is Ion-Exchange Chromatography? - correct answer ✔✔uses a resin to separate substances
according to charge
(+) charged DEAE-Sephadex for anion-exchange
(-) charged phosphocellulose for cation-exchange
even though most proteins negative, center is positive so can still bind cation-exchange resin
, What is Gel Filtration Chromatography? - correct answer ✔✔after both anion and cation-exchange
chromatography, can target different property of proteins = protein size
pass a solution through a "whiffle ball" with varying size holes
small proteins get stuck, large pass around and go further
What is Affinity Chromatography? - correct answer ✔✔Takes advantage of the affinity of different
proteins to certain ligands.
A ligand or other molecule that binds to a protein of interest is covalently attached to the beads in the
column.
protein of interest eluted from column with a solution that disrupts specific binding
elute = remove
supernatant = remaining solution
What is autoradiography? - correct answer ✔✔A process where radiation from a substance is captured
on a camera.
X-ray of gel electrophoresis
What is a densitometer? - correct answer ✔✔A device for measuring the radioactivity in a fragment of
DNA
the darker and denser the band, the more concentration, absorbance
What do labeled tracers do? - correct answer ✔✔detect tiny quantities of nucleic acids or proteins
The number of bases per turn of DNA is___________ - correct answer ✔✔~10.5
What does the Meselson and Stahl experiment confirm? - correct answer ✔✔DNA replication is semi-
conservative
Heavy and light strands with nitrogen isotopes prove
True or False
a. Most biological DNA is positively supercoiled.
b. Triplex DNA is pyrimidine rich
c. Chargaff's rule: A=G & T=C
d. A single zinc finger can recognize 10bp of DNA - correct answer ✔✔A. False, negatively = Left-handed.
Some is right handed = positive
B. True
C. False A=T C=G
D. False, 3 bp, use multiple zinc fingers in a row for more sequence specificity/bp
DNA is not a uniform structure, many localized variants
can substitute protein domains, alpha helix/beta hairpin, in zinc fingers to make hybrids with different
binding specificities (designated by alpha helix) for major grooves of DNA
x-ray crystallography allows us to view and design custom designer DNA-binding proteins = alter gene fxn
in intact cells
In A & Z DNA:
a. Which is right handed helix?
,b. Which one has 12bp per helical turn?
c. Which is the dehydrated form? - correct answer ✔✔a. A
b. Z
c. A
primary DNA is B
Gel electrophoresis - correct answer ✔✔molecular separation technique
can separate nucleic acids or proteins
make AGAROSE gel with slots
hot liquid poured with "teeth"
put DNA in slot, run electric current at neutral pH
DNA = negatively charged (phosphates) = migrates towards positive end
key is friction
DNA stained with fluorescent dye, looked at under UV
unknown DNA electrophoresed parallel with known to determine size
distance migrated = can plot on graph with log of molecular weights or number of bp
ex: 20mm = 910bp
PFGE = Pulsed Field Gel Electrophoresis - correct answer ✔✔for longer sequences
,as large as some chromosomes in yeast
What is PAGE? - correct answer ✔✔Polyacrylamide Gel Electrophoresis
used for PROTEINS
have to denature the protein = treat with detergent, SDS, sodium dodecyl sulfate
SDS-PAGE
SDS advantages:
1) coats polypeptides with (-) charge
2) masks natural charge so results are purely based on molecular weight
What is 2-D gel electrophoresis? - correct answer ✔✔LESS POWER: use two different pH values to
compare, pH affects net charge so different rates of mobility
2-D = 1) proteins electrophoresed through narrow tube gel with pH gradients by ampholytes. Once
protein becomes neutral, no longer moves = isoelectric point
2) gel removed from tube, placed at top of regular gel for SDS-PAGE
What is Ion-Exchange Chromatography? - correct answer ✔✔uses a resin to separate substances
according to charge
(+) charged DEAE-Sephadex for anion-exchange
(-) charged phosphocellulose for cation-exchange
even though most proteins negative, center is positive so can still bind cation-exchange resin
, What is Gel Filtration Chromatography? - correct answer ✔✔after both anion and cation-exchange
chromatography, can target different property of proteins = protein size
pass a solution through a "whiffle ball" with varying size holes
small proteins get stuck, large pass around and go further
What is Affinity Chromatography? - correct answer ✔✔Takes advantage of the affinity of different
proteins to certain ligands.
A ligand or other molecule that binds to a protein of interest is covalently attached to the beads in the
column.
protein of interest eluted from column with a solution that disrupts specific binding
elute = remove
supernatant = remaining solution
What is autoradiography? - correct answer ✔✔A process where radiation from a substance is captured
on a camera.
X-ray of gel electrophoresis
What is a densitometer? - correct answer ✔✔A device for measuring the radioactivity in a fragment of
DNA
the darker and denser the band, the more concentration, absorbance
What do labeled tracers do? - correct answer ✔✔detect tiny quantities of nucleic acids or proteins
