Drybench Plasmid.
Sequencing primers and where they bind
A sequencing read starts from a primer, a short DNA strand that binds the plasmid next to the region to be read; universal primers bind sites that many vectors share.
Reading from a primer
In chain-termination sequencing a DNA polymerase extends the primer along its template (Sanger et al. 1977), so the read starts just past the primer's 3′ end and runs away from it. A primer on the top strand reads clockwise round the map; one on the bottom strand reads anticlockwise.
Vectors were designed with universal primer sites either side of the cloning site, so any insert can be read from both ends (Vieira and Messing 1982; Messing 1983).
Primers on this site
Each primer shipped here is named as its maker's manual names it, because variants share names and differ in bases, and is found exactly at a binding site in a public record before it ships. A site with one mismatch is shown with the mismatch's place counted from the primer's 3′ end; whether the primer works there is not assessed.
See it on a real plasmid
Open pUC19 and find the M13 primer sites either side of its cloning site.
A polymerase copying a template strand from a fixed start: the tour shows RNA polymerase II doing the same with a gene.
References
- Sanger F, Nicklen S, Coulson AR. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A 1977;74:5463-5467. PubMed 271968
- Vieira J, Messing J. The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene 1982;19:259-268. PubMed 6295879
- Messing J. New M13 vectors for cloning. Methods Enzymol 1983;101:20-78. PubMed 6310323
Statements summarised by the site's builder from the references, for scientific review.