| abstract |
The present invention relate to methods and compositions for simultaneously analyzing multiple different polynucleotides of a polynucleotide composition comprising multiple diverse polynucleotide sequences. The subject methods and compositions may also be applied to analyze or identify single polynucleotides; however, the subject methods and compositions are particularly useful for analyzing large diverse populations of polynucleotides, e.g., cDNA libraries. Most embodiments of the invention involve hybridizing terminus probes (of known base sequence) and internal fragment probes (of known base sequence) at adjacent positions on an adapter-modified restriction fragment generated from polynucleotide for analysis, and subsequently joining the terminus probes and internal fragment probes to each other. The terminus probe hybridizes to bases of restriction endonuclease recognition site present at the terminus of a restriction fragment generated from the polynucleotide for analysis. Internal fragment probes hybridizes to the same strand of the restriction fragment that the terminus probe hybridizes to and hybridizes to the restriction fragment portion of adapter-modified representative restriction fragments. The terminus probes and internal fragment probes may be marked so as to facilitate the simultaneous testing of multiple polynucleotides for the presence of many possible nucleotide base sequences. The identity or expression of a particular polynucleotide of interest may be ascertained (or at least partially determined) by producing a short identifier sequence derived from the nucleotide base sequence information obtained from (1) the hybridization of a terminus probe and an internal fragment probe, each of known base sequence, at adjacent positions on a polynucleotide of interest, and (2) the recognition site of a restriction endonuclease used to generate the polynucleotide molecule of interest. Multiple identification sequences may be obtained in parallel, thereby permitting the rapid characterization of a large number of diverse polynucleotides. Parallel processing may be achieved by differentially marking terminus probes or internal fragment probes. Parallel processing may be achieved by using ordered arrays of oligonucleotides that are terminus probes. |