A simple method for screening of NotI linking clones.

Notl linking clones are valuable for ordering of OW fragnents fractionated by Pulsed f ield gel electrophoresis(l) and especially Interested in that they are often associated with transcribable genes(2,3). To faci l i tate the isolation of Notl linking clones, we have devised a method which based on the insertion of Kanamycin cassette bearing Notl sites at both ends into cleavable Notl sites of genome DNA fragrents already cloned Into plasrrrid vectors. The Kanamycin cassette used was constructed by ligation of Notl linker(BamHI-SacII-NotI-Sf1IBamHI: Bceriger mannheim) to Kanamycin Blcck(Kan.Gen.Block: Pharmacia) coimercially obtained. The resultant fragnent was digested with Notl and cloned into Notl site of Bluescript-KS. One of transformants, designated pBS-Kan2, was found to contain the Kanamycin block having heterologous Notl-BamHI fragTents at both ends(Fig. 1A). DNA was extracted from approximately 10000 clones of hunan DM library(Insert length:2-6Kb) in pUC13 vector and digested with Notl. Kanamycin cassette excised with Notl from pBS-Kan2 was ligated to these linear DMA and subjected to transformation of JM103. Analysis of 10 Kanamycin resistant clones, which were randomly picked up, have showed that they Included the human genomic sequences linked to Notl site, inserting Kanamycin cassette into their sites(Fig.lB) and seven of them represented single copy per haploid genome. Furthermore, al l of Notl sites lying them appeared to be methylat1on-free in placenta DMA. Unique site of Sfi l and SacII at each end of Kanamycin cassette wi l l be useful in physical mapping of recontnnant plasmids. I f Kanamycin block 1s cut cut with SacII, i t might be applicable to screen the clones containing sequences around SacII sites which also distributed 1n vertebrate DMA less frequently 1,3).