J Li

Karolinska Institutet, Solna, Stockholm, Sweden

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Publications (7)29.38 Total impact

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    ABSTRACT: The deletion of specific genomic sequences is believed to influence the pathogenesis of certain diseases such as cancer. Identification of these sequences could provide novel therapeutic avenues for the treatment of disease. Here, we describe a simple and robust method called cloning of deleted sequences (CODE), which allows the selective cloning of deleted sequences from complex human genomes. Briefly, genomic DNA from two sources (human normal and tumor samples) was digested with restriction enzymes (e.g., BamHI, BglII, and BclI), then ligated to special linkers, and amplified by PCR. Tester (normal) DNA was amplified using a biotinylated primer and dNTPs. Driver (tumor) DNA was amplified using a non-biotinylated primer, but with dUTP instead of d7TP After denaturation and hybridization, all the driver DNA was destroyed with uracil-DNA glycosylase (UDG), and all imperfect hybrids were digested with mung bean nuclease. Sequences deleted from the driver DNA but present in the tester DNA were purified with streptavidin magnetic beads, and the cycle was repeated three more times. This procedure resulted in the rapid isolation and efficient cloning of genomic sequences homozygously deleted from the driver DNA sample, but present in the tester DNA fraction.
    BioTechniques 11/2001; 31(4):788, 790, 792-3. · 2.40 Impact Factor
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    ABSTRACT: Not I linking clones contain sequences flanking Not I recognition sites and were previously shown to be tightly associated with CpG islands and genes. To directly assess the value of Not I clones in genome research, high density grids with 50 000 Not I linking clones originating from six representative Not I linking libraries were constructed. Altogether, these libraries contained nearly 100 times the total number of Not I sites in the human genome. A total of 3437 sequences flanking Not I sites were generated. Analysis of 3265 unique sequences demonstrated that 51% of the clones displayed significant protein similarity to SWISSPROT and TREMBL database proteins based on MSPcrunch filtering with stringent parameters. Of the 3265 sequences, 1868 (57.2%) were new sequences, not present in the EMBL and EST databases (similarity < or =90%). Among these new sequences, 795 (24.3%) showed similarity to known proteins and 712 (21.8%) displayed an identity of >75% at the nucleotide level to sequences from EMBL or EST databases. The remaining 361 (11.1%) sequences were completely new, i.e. <75% identical. The work also showed tight, specific association of Not I sites with the first exon and suggest that the so-called 3' ESTs can actually be generated from 5'-ends of genes that contain Not I sites in their first exon.
    Nucleic Acids Research 04/2000; 28(7):1635-9. · 8.81 Impact Factor
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    ABSTRACT: Here we describe a new procedure (cloning of polymorphisms, COP) for enrichment of single nucleotide polymorphisms (SNPs) that represent restriction fragment length polymorphisms (RFLPs). COP would be applicable to the isolation of SNPs from particular regions of the genome, e.g. CpG islands, chromosomal bands, YACs or PAC contigs. A combination of digestion with restriction enzymes, treatment with uracil-DNA glycosylase and mung bean nuclease, PCR amplification and purification with streptavidin magnetic beads was used to isolate polymorphic sequences from the genomes of two human samples. After only two cycles of enrichment, 80% of the isolated clones were found to contain RFLPs. A simple method for the PCR detection of these polymorphisms was also developed.
    Nucleic Acids Research 01/2000; 28(2):e1. · 8.81 Impact Factor
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    ABSTRACT: We have partially sequenced more than 1000 NotI linking clones isolated from human chromosome 3-specific libraries. Of these clones, 152 were unique chromosome 3-specific clones. The clones were precisely mapped using a combination of fluorescence in situ hybridization (FISH) and hybridization to somatic cell or radiation hybrids. Two- and three-color FISH was used to order the clones that mapped to the same chromosomal region, and in some cases, chromosome jumping was used to resolve ambiguous mapping. When this NotI restriction map was compared with the yeast artificial chromosome (YAC) based chromosome 3 map, significant differences in several chromosome 3 regions were observed. A search of the EMBL nucleotide database with these sequences revealed homologies (90-100%) to more than 100 different genes or expressed sequence tags (ESTs). Many of these homologies were used to map new genes to chromosome 3. These results suggest that sequencing NotI linking clones, and sequencing CpG islands in general, may complement the EST project and aid in the discovery of all human genes by sequencing random cDNAs. This method may also yield information that cannot be obtained by the EST project alone; namely, the identification of the 5' ends of genes, including potential promoter/enhancer regions and other regulatory sequences
    Gene 12/1999; 239(2):259-71. · 2.20 Impact Factor
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    ABSTRACT: As a step towards developing a new functional test for the identification of tumor suppressor genes, human wild type and mutant RB genes were expressed in the mouse A9 fibrosarcoma cell line under the transcriptional regulation of the tetracycline repressor using two new vectors: pLNCtTA and pETI. Following passage of the transfectants in immunodeficient SCID mice, the wild type RB gene was deleted or functionally inactivated already after the first passage in all 20 tumors tested. In contrast, a non-functional mutant RB gene was maintained in all 10 tumors studied. These results suggest that tests for the identification of tumor suppressor genes may be based on their functional inactivation in vivo, rather than on growth suppression.
    FEBS Letters 06/1999; 451(3):289-94. · 3.58 Impact Factor
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    ABSTRACT: By applying the 'recognition mask' strategy to 300 mammalian sequences containing NotI sites we demonstrated that 5' ends of genes are highly enriched in NotI sites. A NotI linking clone NL2-252 (D3S1678) containing transferrin receptor (TFRC) gene was used as an initial point for chromosomal jumping. One of the jumping clones, J21-045 traverses 210 kbp and links NL2-252 to NL26 (D3S1632), a NotI linking clone containing highly polymorphic sequences. The TFRC gene was mapped to 3q29, close to the telomeric marker D3S2344, by linkage analysis, a panel of hybrid cell lines, GeneBridge 4 panel and FISH. Clone NLM-007 (D3S4302) was found to contain ras-homologous gene RAB7. By FISH and a panel of hybrid cell lines this gene was mapped to 3q21. This region is of particular interest due to frequent rearrangements in different types of leukemia. Clone L2-081 (D3S4283) containing new member of ubiquitin-specific proteases (HAUSP gene) was localized in 3p21 inspiring further investigation of involvement of this gene in development of lung and renal carcinomas.
    FEBS Letters 01/1998; 419(2-3):181-5. · 3.58 Impact Factor
  • Cytogenetics and cell genetics 02/1997; 79(3-4):228-30.