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.
KeywordsAgarose Electrophoresis Fractionation Streptomyces Formamide
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