Inverse sequence-tagged repeat (ISTR) analysis. A novel and universal PCR (polymerase chain reaction)-based technique for genome analysis in the plant and animal kingdom

Abstract

The previously described amplification of a subset of copia-like repetitive sequence elements in the coconut genome by inverse sequence-tagged repeat analysis (ISTR) was extended into a generally applicable strategy for plant and animal genome analysis. A wide range of genomic DNAs was amplified in a polymerase chain reaction in the presence of identical copia sequence-derived primers or primer pairs. The number of loci detected by a single ISTR analysis is large and ranges between 20 and 100, a value which is comparable to that obtained by the AFLP (amplified fragment lenght polymorphism) technique. Depending on the species and the PCR primers, also the number of DNA polymorphisms (at an average 5-50) equals that of AFLP analyses. A modification of the experimental approach allowed the nonradioactive detection of DNA fragments in the sequence gel without membrane transfer thereby allowing for the widespread use of the technology. In addition, specific PCR fragments of interest can be reisolated from the gel, reamplified, sequenced and thus converted for PCR-based sequence-tagged site (STS) analysis