A new method for RAPD primers selection based on primer bias in nucleotide sequence data.

Sequence analysis has proved that decamer nucleotides, used as primers of RAPD (random amplified polymorphic DNA), differ with each other greatly in number of annealing sites in the Arabidopsis thaliana genome. It is called the 'primer bias' by the authors. The biased primers produce a highly variable number of amplicons by polymerase chain reaction (PCR). The number of amplicons is proved to correlate with the number of annealing sites. Therefore, a statistical method is proposed for selecting efficient primers based on the primer bias in the genomic sequence. The method was tested by experiment in A. thaliana genome, and the results demonstrate that the method outperforms routine methods and can substantially increase the efficiency of RAPD methodologies. We also proved that the expressed sequence tags (ESTs) show a highly coincident bias pattern with that of the whole genomic sequence, and can therefore be used to assess efficiencies of primers for species whose genomic sequence data are currently unknown.