The Use of Short Adapters for Priming PCR of Unknown Chromosomal DNA Fragments
For the analysis of unknown stretches of deletable DNA from Streptomyces lividans 1326 [3,4], we plan to apply the following protocol for substractive hybridization : randomly sheared substracter DNA from the variant strain Nl (with the respective DNA deleted) is biotinylated , mercurated  and hybridised in great surplus with unlabelled wild-type DNA fragments generated by a Sau3A partial digest. Subsequent affinity chromatography on a Thio-propyl-Sepharose 6B (Pharmacia) and Streptavidin-Agarose (Gibco-BRL) double column removes all DNA hybrids containing at least one strand of labelled DNA, leaving a solution with a small amount of wild-type specific fragments, each only in a few copies. Thus, prior to further studies, these fragments have to be amplified considerably.
KeywordsStreptavidin Agarose Double Column Preliminary Positive Result Short Adapter Pharmacia Column
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- 1.Bjourson AJ, Cooper JE (1988). Isolation of Rhizobium loti strain-specific DNA sequences by substractive hybridisation. Appl Env. Microbiol 54: 2852–2855.Google Scholar
- 5.McInnes JL, Vise PD, Habili N, Symons RH (1987). Chemical biotinylation of nucleic acids with photobiotin and their use as hybridisation probes. Focus 9–4:1–3.Google Scholar
- 6.Schneider J, Kutzner HJ (1989). Distribution of modules among the central region of the genomes of several actinophages of Faenia and saccharopolyspora. J Gen Microbiol 135: 1671–1678.Google Scholar