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Plasmids and Gene Insertion
Name: Sandra P.
Status: Educator
Age: 50s
Location: N/A
Country: N/A
Date: 2001
Question:
What criteria are used for selecting plasmids to use in
sequencing experiments? I was in a lab this summer where the researcher
was using an artificial plasmid to try to sequence part of Drosophila's
genome (to check the released results). She was unhappy with how it was
working, but did not explain what she meant by "this is the best I can
get".
I also did not understand why she was trying to get the gene to
insert both forward & backwards.
Replies:
A plasmid is a vehicle that can carry artificially
inserted DNA. It will replicate in E. coli, and with
its own replication it will also replicate the
inserted DNA, independent of it's origin. In a way one
can see a plasmid as a minute DNA factory. The main
criteria for a 'good' plasmid is that it takes up the
insert you want to put in, and that it replicates in
sufficient amounts,and that it does not destroy your
insert during the process. There are limiatations to
each of these steps: large inserts require specialized
plasmids (cosmids or YACs for megabase sized-
inserts), the larger a plasmid+insert, the lower its
replication rate, but there are ways to improve the
yield, and certain plasmids result in frequent
deletions of (parts of) the insert, although this is
sometimes due to the host (the E. coli or another host
cell) or due to the nature of the insert as well.
On paper the ideal experiment can easily be drawn. In
real life we have to accept sometimes that we cannot
get the system to work ideally. It is frustrating but
sometimes one has to accept 'this is the best I can
get'. I cannot judge if that was true in the case you
experienced.
Your second question is why an insert has to be
present in two directions. When one wants to determine
the DNA sequence of an insert, it is not necessary to
have it cloned in two orientations, but it is advised
to sequence it from both ends, ideally to sequence
both strands of each insert completely. Instead of
'turning the insert around' (which means cloning it in
2 directions) one can make use of a sequence primer
that matches the other strand, so that the sequence
reaction starts on the other end of the insert.
Sometimes this is the only option, when for instance
an insert works out to be toxic for the cell in one
particular orientation. Such toxicity is often due to
translation of the insert, whereby toxic peptides are
produced. In the other direction the insert wouldn't
be translated, and thus wouldn't be toxic.
As a note for completion: inserts are not only cloned
into vectors for sequencing. There may be other
reasons why a particular fragment has to be cloned,
and then sometimes it is essential to get the insert
in both directions.
Dr. Trudy Wassenaar
Molecular Biologist
While there are only a couple essentials that a plasmid must have, DNA
workers have engineered a number of features into many plasmids that make
them easier to use. First the essentials: 1) it must have the _ori_ gene,
which enables the plasmid to be replicated in the host cells, 2) it must have
a "selectable marker" so that only cells that have the plasmid will grow in
culture. This is almost always a gene that confers resistance to a particular
antibiotic, so that cells grown in the presence of the drug will all contain
the plasmid.
Other "nice" features include a second selectable marker, perhaps
another
antibiotic resistance gene, or more commonly, the beta-galactosidase gene,
which allows a research to know, just by looking at a colony on a petri dish,
if the plasmid contains foreign DNA inserted into it. There is also usually a
cluster of sites for up to two dozen restriction enzymes, so that workers can
clone DNA more easily into the plasmid. Lastly, and pertaining to your
question about sequencing, it contains a stretch of about 12 to 20 bases (the
primer site) just upstream from the cluster of restriction sites. As part of
the sequencing process, one uses the primer site and reads sequence into the
inserted region of foreign DNA.
Sequencing only tells you the sequence of one strand of the
double-stranded DNA. To be certain you didn't read an occasional base in
error, you generally want to sequence both strands of the DNA to obtain
complementary sequences. To do this, the researcher wanted to have the
inserted DNA present in both orientations in the plasmid. After sequencing
two different plasmids from the same primer site, each one with the insert in
a different orientation, one would have the complete sequence of both strands.
Actually, most researchers don't use this approach of obtaining two
different plasmids with the insert in opposing orientations. An easier way is
to sequence the complementary strand of DNA by using a second primer site at
the other end of the inserted DNA. So, in a way, you are sequencing the
insert from "left to right" and then again from "right to left." There are,
however, particular situations which require the approach your researcher was
using.
Paul Mahoney, Ph.D.
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Update: June 2012
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