Not I ̄ anking sequences : a tool for gene discovery and veri ® cation of the human genome

A set of 22 551 unique human NotI ̄anking sequences (16.2 Mb) was generated. More than 40% of the set had regions with signi®cant similarity to known proteins and expressed sequences. The data demonstrate that regions ̄anking NotI sites are less likely to form nucleosomes ef®ciently and resemble promoter regions. The draft human genome sequence contained 55.7% of the NotI ̄anking sequences, Celera's database contained matches to 57.2% of the clones and all public databases (including non-human and previously sequenced NotI ̄anks) matched 89.2% of the NotI ̄anking sequences (identity >90% over at least 50 bp, data from December 2001). The data suggest that the shotgun sequencing approach used to generate the draft human genome sequence resulted in a bias against cloning and sequencing of NotI ̄anks. A rough estimation (based primarily on chromosomes 21 and 22) is that the human genome contains 15 000±20 000 NotI sites, of which 6000±9000 are unmethylated in any particular cell. The results of the study suggest that the existing tools for computational determination of CpG islands fail to identify a signi®cant fraction of functional CpG islands, and unmethylated DNA stretches with a high frequency of CpG dinucleotides can be found even in regions with low CG content. INTRODUCTION Draft sequences of the human genome were recently reported (1,2). Much work remains to be done to produce a complete ®nished sequence and progress can be best assured by a diversity of approaches (1). At present, one of the principal goals for genome research is a careful and systematic validation of the assembled sequence (1±4). In this respect it becomes critically important to develop and to apply strategies capable of ful®lling this crucial aim. These strategies should exploit approaches independent of those used to generate the original genome sequence. Short sequences ̄anking the rare restriction sites, for instance NotI, might serve as a tool for validation of human genome structure. NotI linking clones contain pairs of sequences ̄anking a single NotI recognition site, while NotI jumping clones contain DNA sequences spanning between neighboring NotI restriction sites. Such clones were shown to be tightly associated with CpG islands and genes (5,6). The use of NotI linking and jumping clones as framework markers was proposed to de®ne the structure of large regions of human chromosomes (7±13). To achieve this goal, simpli®ed procedures for the construction of NotI jumping and NotI linking libraries were developed and a number of chromosome 3-speci®c and other chromosome-speci®c and total human NotI linking libraries were prepared (7±15). One thousand human chromosome 3-speci®c NotI linking clones were partially sequenced (6). Among these, 249 unique clones were identi®ed and 152 were carefully analyzed. To localize these clones, PCR, Southern hybridization, pulsed ®eld gel electrophoresis (PFGE) and twoor three-color ̄uorescent in situ hybridization (FISH) were applied. In many *To whom correspondence should be addressed at: Microbiology and Tumor Biology Center, Karolinska Institute, Box 280, 171 77 Stockholm, Sweden. Tel: +46 8 728 6750; Fax: +46 8 319 470; Email: eugzab@ki.se The authors wish it to be known that, in their opinion, the ®rst two authors should be regarded as joint First Authors +AQ936570±AQ939834, AJ322533±AJ343893 ã 2002 Oxford University Press Nucleic Acids Research, 2002, Vol. 30 No. 14 3163±3170