Published primers were compiled from the references given in the list below.
Their sequences were searched on the tobacco (Nicotiana tabaccum L.) sequence in GenBank (locus ID CHNTXX, Acc. No. Z00044 and S54304) with the help of Omiga 1.1.3 software (Accelrys, Oxford, U.K.).
Sequences that did not match closely enough to the tobacco sequence were omitted [depending on demand, dedicated monocot and conifer versions may follow; [please indicate your interest!].
The primer situations were added as 'features' to a number of tobacco chloroplast sequence files (available on request) and graphics were drawn with Omiga software.
Primer designations from the original publications were retained as much as possible. Sometimes, the name of the first author or some other hint were included in the primer names. 'F' and 'R' are the direction (forward or reerse) of the primer relative to the toacco sequence (some authors have named their primers on the basis of the direction of transcription, which can be a source of confusion here).
New primers were developed to fill remaining gaps, or to replace primers with poor performance in our lab. This was done by comparing and aligning chloroplast DNA sequences available in GenBank by eye, with the help of Omiga, BLAST (Altschul et al. 1997), or a suite of DOS programs written by John Antoniw (1995).
Sufficiently conserved potential primer sites were visually checked for anormalities like biassed GC/AT percentages, mononucleotide stretches, or apparent palindromes; site with such features were avoided whenever possible.
Additionally, the primer sequences were BLASTed against the complete chloroplast genome sequences of Atropa belladonna, Spinacia oleracea, Arabidopsis thaliana, Populus trichocarpa (Heinze et al. unpublished), Oryza sativa, Pinus thunbergii, and Marchantia polymorpha (as retrieved from GenBank early July 2005). This was done using BLASTALL (available from NCBI). The position of the 5' nucleotide of the primer in these complete chloroplasts is indicated in the primer table. The orientation of the primer in tobacco is indicated by an 'F' (forward) or an 'R' (revers). With these data, it is easily possible to calculate PCR fragment sizes, and to estimate expected sizes from 'new' taxa (Grivet et al. 2000)