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Title: The application of positional cloning techniques to a region on chromosome 3 deleted in small cell lung cancer
Author: Cahn, A. P.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 1997
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The 3p1.2-p1.3 chromosomal region has a dark Giemsa staining pattern and in accordance with this, "rare cutter" mapping has shown the YAC clones from the region to display a paucity of "rare cutter" restriction enzyme sites. Only thirteen putative CpG islands and five Not I sites were identified in the entire deleted region. Transcription mapping of selected loci from this region was performed using YAC clones and cosmids from the deletion using three approaches:- 1) Selected YAC clones were used individually or as pools directly as hybridisation probes on to cDNA libraries. These included human bronchial epithelium, foetal brain and placental cDNA libraries. Four cDNA clones were isolated and analysis demonstrated the presence of sequences deleted from U2020 genomic DNA. These clones were further investigated by sequencing and hybridisation to Southern and Northern blots. 2) Since 12% of Not I sites are independently associated with transcribed sequences and up to 90% lie in CpG islands a YAC from the region containing a Not I site was isolated and the DNA around this site subcloned. 3) Cosmids from the deleted region were isolated by hybridisation of YACs and unique sequence probes, from the D3S3 locus at 3p1.2-p1.3, to a gridded flow-sorted chromosome 3 cosmid library. Using these techniques 1.4 Mb of cloned DNA from the deleted region was scanned for transcribed sequences. No genes were identified and only repetitive elements isolated. This is consistent with this region of chromosome 3 being gene poor and repeat rich. For gene isolation in gene poor regions a more targeted approach, such as cloning around CpG islands, may be more effective than methods designed for transcribed sequence scanning, such as exon amplification or direct cDNA selection.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available