|prev page||PLNT3140 Introductory Cytogenetics
Lecture 17, part 3 of 4
m1 4cM d 1 m2
During the initial walk, you have to walk in both directions until you can determine which is the right direction. Often, directionality can be determined if one of the overlapping clones detects a polymorphism. This facilitates a 3 point cross with the probe, m2 and d. The other way of determining directionality is to keep walking until you find a clone that hybridizes with m1.
Making an end-specific probe. BAC
is digested with a restriction enzyme, such as Alu1, that cuts
frequently, yeilding small fragments. (Note
that the insert is much bigger than the BAC vector that
A primer specific for one of the
BAC sequences bordering the cloning site is added, along with
polymerase and labeled nucleotides. The primer is
elongated, but elongation only proceeds as far as the next
at most a few hundred nucleotides downstream. Thus, the only
the BAC insert to be labeled is at one end of the insert.
If you user a primer synthesized
match the region of the vector immediately flanking the
labeling reaction will proceeed into the insert, and terminate
end of the fragment, where AluI cut. One of the advantages of
approach is that the same primer can be used for all clones,
all inserts are in the same vector!
From this point on, every gene is a special case.
Complementation is the method of choice for experimental systems that allow transformation. For example, disease resistance genes in plants have been cloned by transforming susceptible plants with DNA from each BAC in a contig, and screening for resistant plants.
cDNA screening is an approach that sometimes works. eg. screening for a disease gene in humans:
(Figure from http://www.gdb.org/Dan/fig9.html)
Once you have an ordered library:
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