Optimal Primer Design for Sequencing in E. coli
Agencourt has designed new primers for their CopyControl vector series which use 3' ends which are low frequency in the E. coli genome. The two 7-mers are GTCTAGG and CTAGGAA which occur 3 and 7 times in the genome, respectively, and have near 50% GC content. The 7-mer GCCTAGG does not occur in the genome, but was rejected due to self-complementarity. They chose an 8-mer which was unique, and the two primers they finally chose for their sequencing vector were GTA CAA CGA CAC CTA GAC and CAG GAA ACA GCC TAG GAA (Note that their final choice is inconsistent with their reported plan). The low frequence of the CTAG 4-mer appears to be the key insight, and this might be useful in designing novel primers and primer binding sites. See Epicentre Forum Vol 13(1) p 17, "An improved CopyControl fosmid vector maximizes end-sequencing results.
At the MIT biopolymers facility and probably with most sequencing centers, when doing a run-off sequencing reaction, an extra (sometimes pretty high amplitude) 'A' peak is seen at the end before the template ends. The workers at the biopolymers facility seemed surprised when they were told about this, but they were able to find out that the sequenase enzyme used in the sequencing reaction is a genetically modified form of Taq. Therefore, it is most probable that the extra A is the template-independent A that Taq tends to add to the 3'-end of DNA.
Q: The MIT sequencing center recommends an amount of template DNA for a sequencing reaction. If I have a mixed sample of DNA (for example, two plasmids purified from a culture) and want some of it sequenced (for example, one of the plasmids), should I submit the recommended amount of total DNA or the recommended amount of the DNA of interest?
A: It's best to submit a pure sample...but if you cannot, I would make sure that you submit the required amount of the template that the primer that you are using will anneal to.
Any other DNA will not get labeled with the fluorescent dyes that we use to call a sequence....and will hopefully be inert...it should be inert unless your primer also anneals to the contaminant DNA.
The dynamic range of the DNAsequencer is between 10 to 1000 nanograms...so if you are in that ballpark it should work.