3' transcript mapping of the Chlamydomonas reinhardtii chloroplast psbK mRNA using a novel method to prepare 3'...

We have developed a novel method for the rapid preparation of large quantities of 3' end-labeled single-stranded (ss) DNA (ssDNA) probes for transcript mapping. A recombinant phagemid vector containing the probe sequence was used to raise large quantities of ssDNA. Based on the DNA sequence of the probe, an oligonucleotide primer complementary to the ssDNA probe was synthesized. Annealing of the primer to the purified ssDNA phagemid clone produced a short double-stranded DNA duplex containing a restriction site, which was then cut with a restriction enzyme to generate a 5' overhang of the primer strand. The phagemid DNA was labeled at the 3' end with the Klenow fragment of polymerase I. The low melting temperatures of the short primer-phagemid duplex caused the primer to dissociate during the hybridization of the probe to algal RNA. For this reason, the probe can be used for S1 mapping without further purification. This method was used to map the 3' end of the Chlamydomonas reinhardtii chloroplast psbK transcript.