Primer orientation, forward and reverse - (Apr/20/2007 )
I'm working at sequencing the 16s rRNA gene for acidophilic bacteria. I'm using universal primers such as 27f and 1492r for my PCR reactions and a few others (530r, 1100r, 782r, and 765f) in order to sequence them. I've gotten a few sequences back and want to piece them together into a 'contig' (is that the right term?).
My question is if anyone knows how these primers are oriented in the original E. coli sequence? And does anyone have any suggestions on literature for piecing together sequences into a full gene?
The F and R refer to the direction of the primer. The number is the approximate location of the primer on the 1500 bp 16S sequence. See the papers referred to here: http://openwetware.org/wiki/Bacterial_species_identification
I'm just wondering, is the number where the primer ends and the amplified sequence begins? So for example with 27f, the primer is so many positions before position 27 and your amplified sequence is then position 28 on?
PCR products always directly incorporate the primer. So, the product will start with the 5' end of the primer. Sequencing results, however, will start around 10 bp downstream from the 3' end of the primer (usually). The locations of these primers is only approximate, both because people are sloppy in their definitions of the numbering, and because different species have different lengths of some portions of the 16S sequence. With modern sequencing capability, it is likely that a complete 16S sequence could be obtained by PCR with 27f and 1492r, followed by sequencing the PCR product with those same two primers. For reliability and publication, one normally likes to sequence a fragment in both directions, however, and the internal primers are useful for that purpose.
Thanks, that's very helpful. I've actually gotten my first sequences back and have aligned them into a complete contig. So I'm well on my way!