BCH5413 EXAM 3 ACTUAL FINAL STUDY PAPER 2026 COMPLETE QUESTIONS AND ANSWERS

BCH5413 EXAM 3 ACTUAL FINAL STUDY PAPER
2026 COMPLETE QUESTIONS AND ANSWERS


◉ Southern Blot analysis: preparation for transfer of DNA
- to increase efficiency...
- so far.... Answer: - to increase the transfer efficiency of large DNA
fragments, soak the gel in dilute HCl to partially depurinate the DNA
(DNA will be cleaved at site of depurination)
- denature the dsDNA by soaking gel in dilute NaOH (dsDNA will
convert to ssDNA)
- neutralize the pH of gel by soaking in solution buffered to pH 7


So far: DNA fragments have been separated by size, denaturing the
DNA fragments will allow the fragments to be identified according to
sequence


◉ Southern blot analysis: transfer of DNA. Answer: - dish with buffer
- solid support
- wick/watman paper layed on top of support and ends dip into
buffer
- agarose is above wick paper
- nylon membrane above agarose

,- membrane traps and binds nucleic acids from the gel
- blotting paper above membrane (cappilary action causes buffer to
move into blotting paper)
- weight on top of blotting paper


◉ Southern blot analysis: ready to probe. Answer: (After transfer of
DNA)
- membrane contains bound fragments of ssDNA which retain the
pattern of size separation
- "fix" DNA to blot by baking or UV light
- "block" the blot by incubating with solution containing BSA to
decrease non specific binding
- "probe" the southern blot by allowing a labeled fragment f ssDNA
to anneal to its complementary sequence on the membrane


◉ Southern blot analysis: preparation of probes by random priming.
Answer: Random primer: oligonucleotide 6-10 nucleotides in length
in which each position can be any of the four bases
- therefore a 6 base oligo would have 4096 unique types of oligos in
a representative sample


*random oligonucleotide primers anneal to template DNA
- denature with heat to allow hybridize ssDNA of probe to ssDNA
bound to solid support

,◉ Southern blot analysis: what klenow does to dCTP. Answer:
klenow cleaves off diphosphate and incorporates dCTP into DNA
probe


◉ Southern blot analysis: klenow fragment & nucleotides. Answer:
large fragnment of DNA Pol 1


nucleotides:
- dGTP
- dATP
- dTTP
- radioactive labeled dCTP


◉ Southern blot analysis: preparation of probes by end labeling.
Answer: - ds oligonucleotide matches sequence of interest
- treated with calf intestine alkaline phosphatase
- incubate de-phosphorylated oligonucleotide with T4 kinase +
radioactive ATP
- T4 kinase incorporates ATP into probe


*need to denature dsDNA probe to hybridize to ssDNA on membrane

, ◉ Southern blot analysis: preparation of probes by non radioactive
labeling. Answer: - dUTP is used instead of radioactive dCTP
- dUTP is labeled with DIG (highly immunogenic; high affinity to
antibodies and generate strong signal)


*after hybridization of DIG-labeled prove you can incubate with anti-
DIG antibody linked to an enzyme that converts substrate to light;
light can be detected


◉ Southern blot analysis: hybridization and detection
- hybridize and wash. Answer: Hybridize: allows the denatured,
labeled DNA probe to anneal to DNA on the solid support (nylon
membrane)


Wash: remove probe that is nonspecifically bound to the solid
support


*conditions should be carefully adjusted to minimize chance of non
specific binding
**need to maximize signal-to-noise ratio


◉ Southern blot analysis: hybridization and detection
- steps. Answer: - incubate labeled ssDNA probe
- heat DNA probe so dsDNA melts