SOLID-PHASE CLONING TO CREATE SUBLIBRARIES SUITABLE FOR DNA-SEQUENCING

A solid-phase method is described to create subclones, suitable for DN A sequencing, from lambda or cosmid libraries. The purified target DNA is sonicated and two linkers, with one oligonucleotide biotinylated i n the 5'-end, are ligated to the ends of the fragments produced by son ication. After size separation, the fragments are immobilised onto a s olid support and the non-biotinylated strand of each immobilised fragm ent is eluted. In this way, a library of single-stranded fragments is obtained. All fragments contain 'universal' flanking sequences of 22 b ases introduced by the linker ligation. These flanking sequences can s ubsequently be used for solid-phase cloning into a single-stranded vec tor containing the complementary sequences. Thus, cloning can be achie ved without the use of ligase or restriction enzymes. The resulting su bclones are used for direct solid-phase sequencing and the immobilised strand can be used to selectively remove homologous DNA from the libr ary of single-stranded fragments. Thus, a sublibrary of non-sequenced fragments can be created. Here, we show that a library of clones, suit able for direct solid-phase sequencing, can be obtained starting with lambda DNA. The efficiency of selective hybridisation of homologous an d non-homologous fragments was investigated. The possibility of using this approach for automated cloning strategies for large-scale genomic and cDNA sequencing is discussed.