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Electrophoresis Exam Questions With Solutions (A+)
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Electrophoresis Exam Questions With Solutions (A+)Basic DNA Chemistry Right Ans - -Negatively charged because of sugar
phosphate backbone
-Made up 4 base pairs,
-has 3' and 5' ends, strands run antiparallel to each other
-bases are bound to each other through hydrogen bonding
-DNA is extended at the 3' OH end
How does DNA electrophoresis work? Right Ans - -There is an electric field
and DNA is run along a polysaccharide gel. Because of the negative charge on
DNA from the sugar phosphate backbone, the DNA is attracted to the anode
and repelled by the cathode. The fragments travel though pores in the gel, and
separate based on their size. Shorter fragments travel farther and faster.
What is DNA electrophoresis used for? Right Ans - -DNA Sequencing
(Sanger or Gilbert methods/ capillary)
-Genetic Disease diagnosis
-Gene Expression studies
-Forensic Testing
-Paternity Testing
How does separation take place? Right Ans - -Fragments must weave
through pores in the gel which slows them down based on their size
-Shorter molecules travel further than longer molecules as they are pulled
toward the anode
-Linear DNA migrate inversely proportional to the log10 of their weight
What does the migration of DNA depend on? Right Ans - -Dependent on
length of the molecules AKA molecular weight of fragment, DNA has a "linear"
mass to charge ration that is nearly perfect, so length of fragment is the only
factor
Agarose vs. Polyacrylamide Gel Right Ans - -Agarose gel is used more for
qualitative analysis whereas Polyacrylamide is for quantitative analysis
-Agarose is primarily used for DNA electrophoresis, but if you want superior
resolution than polyacrylamide gel can be used (1 nucleotide resolution)
, What is DNA sequencing? Right Ans - -a method developed by Sanger and
Gilbert to determine the exact nucleotide sequence of DNA fragments
-traditional methods (Sanger) utilize defective bases (missing 3' OH group) in
a DNA sequencing reaction to terminate DNA synthesis when added, separate
reactions for each well using one of the four deceptive bases
-mixture of regular and defective bases used, yields many different sized
fragments depending on when the defective base is added
-perform electrophoresis with 4 lanes, one for each defective bases, length of
fragment tells you where the defective base was added, read all four lanes to
find the whole sequence
What is the difference between the traditional Sanger method and automated
sequencing? Right Ans - -Sanger is much slower, utilizes a gel
electrophoresis with separate lanes for each defective base
-first method tagged fragments with radioactive primers
-develop X-ray film over the gel to view how far each fragment traveled
-manual version (by hand)
-Automated sequencing, utilizes capillary electrophoresis
-tags defective bases with fluorescent markers, all fragments are run in the
same lane
-laser interrogates the bases, emission is received by a detector, each base is
labeled with a different color
-much faster but only works well for fragments up to 1000 base pairs because
the speed of migration is not far enough apart for the fluorescent detector to
tell the difference between 1000 and 1001, based on the inverse log10.
What is Shotgun sequencing? Right Ans - -if a fragment is over 1000 bases
long it must be cut randomly into smaller fragments using restriction enzymes
-each fragment is then cloned in a separate bacterial vector and sequenced
-computer looks for overlaps in each sub fragment and pieces the DNA back
together
-fastest method to sequence large amounts of DNA
What is capillary electrophoresis? Right Ans - A method of separation of
ions based on electrophoretic mobility
-Tubes have high surface to volume ratio which gives the tube the ability to
radiate heat quickly
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