BCH5413 Exam 3 Final Study Paper Questions and Answers Verified Solutions Latest Update 2026/2027

BCH5413 Exam 3 Final Study Paper
Questions and Answers Verified Solutions
Latest Update 2026/2027

Question:
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


Question:
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

,Question:
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


Question:
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


Question:
Southern blot analysis: what klenow does to dCTP.

Answer:
klenow cleaves off diphosphate and incorporates dCTP into DNA probe


Question:
Southern blot analysis: klenow fragment & nucleotides.

Answer:

,large fragnment of DNA Pol 1
nucleotides:
- dGTP
- dATP
- dTTP
- radioactive labeled dCTP


Question:
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


Question:
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


Question:
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


Question:
Southern blot analysis: hybridization and detection
- steps.

Answer:
- incubate labeled ssDNA probe
- heat DNA probe so dsDNA melts
- allows for labeled DNA probe to hybridize to complementary DNA
strands on membrane
*then you must wash
*hybridization can be done in a tube or in a plastic bag for incubation


Question:
Southern blot analysis: stringency.

Answer:
Stringency: properties f hybridization conditions that dictate the degree of base mismatch permitted
between probe and target DNA. Low stringency permits greater mismatch and may result in high
background or "noise" while high stringency permits less mismatch but may result in reduced
signal


Question:
Southern blot analysis: how to increase stringency.

Answer:
- decrease [Na+] (ioninc strength)
- increase hybridizatoin and wash temperatures
- increase duration of wash


Question:
Southern blot analysis
- cDNA probes with different levels of species similarity.