Gene Cloning

362 Gene Cloning
Q11.3. Why is it essential to remove all DNA from the RNA sample before
northern analysis?
A11.3. The labeled probe used to detect the RNA during northern analysis
will also hybridize to any contaminating DNA as all genes, expressed or otherwise,
will be present in the genomic DNA; this can give you a false positive
for transcription of the gene.
Q11.4. The QRT–PCR protocol utilizes random hexanucleotide primers to
generate the cDNA prior to QRT–PCR. What is the advantage of using random
hexanucleotides rather that a gene-specific primer?
A11.4. Random hexanucleotides will generate a population of cDNAs
reflecting the range of RNA molecules in the starting sample. It is therefore
possible to subsequently use quantitative real-time PCR on the same mixture
of cDNAs to determine the levels of mRNA for any gene for which the DNA
sequence is known.
Q11.5. Which technique, standard northern or reverse northern analysis
would be best to study (a) the levels of mRNA for the same gene in several
RNA samples (e.g. from different cell types) or (b) the levels of mRNA for
several genes in the same RNA sample?
A11.5. (a) Standard northern analysis is best suited to determining the
mRNA levels for the same gene from several samples, because each RNA sample
can be immobilized on the same membrane and the same labeled probe
hybridized and the level of hybridization quantified. You cannot use two different
probes in the same hybridization, so cannot analyze more than one
gene at a time.
(b) Reverse northern would be best for determining the mRNA levels of several
genes within the same sample because the probes are immobilized on
the membrane, so several probes can be immobilized on the same membrane.
The labeled cDNA will then hybridize to the immobilized probe and
the level of hybridization of each mRNA can be quantified.
Q11.6. Figure 11.15b shows how a series of overlapping fragments can be
used to map where a transcription factor binds within a large DNA fragment.
Why would you use overlapping fragments rather than a series of
non-overlapping fragments?
A11.6. If you used a series of non-overlapping fragments, the DNA binding
site may be spilt between two adjacent fragments and therefore no binding
would be detected. For example, if you have two fragments 1–40 and 40–80,
the protein may require sequences from 35 to 50 to bind, and so would bind
neither fragment. Overlapping fragments are used to ensure that any potential
site is present on at least one fragment. For example, if you have three
fragments 1–40, 20–60 and 40–80, a protein that binds from 35 to 50 could
bind to the 20–60 fragment.