|A data base for PCR primers in the chloroplast genome|
The database presented here has been compiled on the basis of information in numerous publications, and especially by communications from Bill Hahn, Delphine Grivet, Remy Petit, and Keith Livingstone. 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' for 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 drection of transcription, which can be a source of confusion here). Additionally, the first or the last nucleotide for each primer in the tobacco sequence is given (the first one for 'F' and the last for 'R' primers). With these data, it is easily possible to calculate PCR fragment sizes, and to estimate expected sizes from 'new' taxa (Grivet et al. 2000).
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.
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