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Name: Sandra P.
Status: Educator
Age: 50s
Location: N/A
Country: N/A
Date: 2001

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.

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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