Use Of The RAPD Assay For The Detection Of DNA Mutations Of Ultraviolet-induced Streptomyces Spp. AP19-1

Randomly amplified polymorphic DNA (RAPD) analyses involve the amplification of anonymous segments of genomic DNA by PCR techniques using oligonucleotide primers constructed in the absence of any knowledge about the target DNA sequence. But the resolution and repetition is low because of the low annealed tempture. In order to increasing the efficiency of the technique of RAPD, we determined the optimal RAPD amplification conditions to obtain a rapid and accurate system for streptomycetes. The following parameters were modified: template DNA, DNA polymerase, MgCl₂, dNTP and primer concentration; the ramp time from annealing to extension, temperature of annealing and the number of thermal cycles. We obtained the following optimization, reliable and reproducible RAPD patterns:each assay (20μl volume) contained incubation buffer, 3.0mM MgCl₂. 400μM dNTP, 0.4μM primer, 2U Taq DNA polymerase and 200ng template DNA.The PCR program was as follows: initial denaturating step at 94 ℃ for 5 min,fowllowed by 40cycles of 94℃ for 30 sec, annealing at 36.4℃ for 30 sec,extinction at 72℃ for 2min. Finally, extension at 72℃ for 10min. Thereinto, the ramp time from annealing to extension was 108 sec. We had obtained a UV irradiated stretomyces spp. strain whose antibiotics-producing ability were over the original strain. 50 primers were examined as to their applicability in clarifying the genetic background for such mutants using RAPD analysis. As a result, we obtained two specific fragments that are 480bp and 570bp respectively. Then those fragments were extracted and sequencd. The result of the DNA sequence alignment in NCBI showed that the 480bp fragment had 100 persent homology with the intergenic region of the pathogenicity island of a shiga toxin producing Escherichia coll It was presumed that the sequence was involve in the regulation network of the antibiotics metabolite of streptomyces spp.