Solid-phase cloning to create sublibraries suitable for DNA sequencing.
A solid-phase method is described to create subclones, suitable for DNA sequencing, from lambda or cosmid libraries. The purified target DNA is sonicated and two linkers, with one oligonucleotide biotinylated in the 5'-end, are ligated to the ends of the fragments produced by sonication. After size separation, the fragments are immobilised onto a solid support and the non-biotinylated strand of each immobilised fragment is eluted. In this way, a library of single-stranded fragments is obtained. All fragments contain 'universal' flanking sequences of 22 bases introduced by the linker ligation. These flanking sequences can subsequently be used for solid-phase cloning into a single-stranded vector containing the complementary sequences. Thus, cloning can be achieved without the use of ligase or restriction enzymes. The resulting subclones are used for direct solid-phase sequencing and the immobilised strand can be used to selectively remove homologous DNA from the library of single-stranded fragments. Thus, a sublibrary of non-sequenced fragments can be created. Here, we show that a library of clones, suitable for direct solid-phase sequencing, can be obtained starting with lambda DNA. The efficiency of selective hybridisation of homologous and non-homologous fragments was investigated. The possibility of using this approach for automated cloning strategies for large-scale genomic and cDNA sequencing is discussed.
Hultman
T
T
Royal Institute of Technology, Department of Biochemistry, Stockholm, Sweden.
Uhlén
M
M
D001483:Base Sequence; D001709:Biotechnology; D003001:Cloning, Molecular / Q000379:methods*; D004269:DNA, Bacterial / Q000235:genetics; D004277:DNA, Single-Stranded / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015723:Gene Library; D006801:Humans; D008280:Magnetics; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D017422:Sequence Analysis, DNA / Q000379:methods*
D004269:DNA, Bacterial; D004277:DNA, Single-Stranded