摘要：

In this study, we report a simple procedure for developing and using a new type of polymerase chain reaction (PCR) primers, named 'high frequency oligonucleotides - targeting active genes (HFO-TAG)'. The HFO-TAG primers are constructed by first using a "practical extraction and report language (Perl)" script to identify oligonucleotides (8, 9, and 10 base) that exist in high frequency in a 4,700 EST-unigene watermelon fruit library. This computer-based screening yielded 3,200 oligonucleotides that exist 32 to 335 times in 4,700 EST-unigenes constructed for watermelon. Of these, 192 HFO-TAG primers (present 51-269 times in the 4,700 EST-unigenes) were used to amplify DNA from closely related watermelon cultivars. The average number of DNA fragments produced by a single HFO-TAG primer among the watermelon cultivars was considerably higher than the number of fragments produced by inter-simple sequence repeat (ISSR) or randomly amplified polymorphic DNA (RAPD) primers. Also, the HFO-TAG primers produced considerably more fragments than the ISSR or RAPD primers from a watermelon cDNA library that was used as a template. These results indicate that the HFO-TAG primers should be more specific in targeting active gene loci. The extensive EST database available for a large number of plant and animal species should be useful for developing HFO-TAG primers that can be used in genetic mapping and phylogenic studies of important crop plants and animal species.

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