Comparison of the positional cloning methods used to isolate the BRCA1 gene

Abstract

A critical step in positional cloning is the identification of candidate genes from a large, genetically defined region. Candidate gene isolation by hybrid selection, genomic sequencing, and direct cDNA library screening identified 45 candidate gene fragments (CGFs) from a 600 kb genomic region that contains the BRCA1 gene. These CGFs define a minimum of 15 genes, six of which are newly localized to the BRCA1 region. We present an analysis of the efficiency and the sequences generated for each of these methods. We also compare our CGF set to those reported for the BRCA1 region by three other groups, revealing a surprising lack of overlap among the sets.

Complete genomic sequence and analysis of 117 kb of human DNA containing the gene BRCA1.

“…The high number of EST sequences found to map to the VAT1 gene suggest that it is an abundant message. Two of the ESTs map to the first intron of this gene (Harshman et al 1995 rpL21: A pseudogene of ribosomal protein L21 was identified in intron 13 of the BRCA1 gene (Figs. 1 and 6).…”

“…Two of the cDNA sequences (GenBank accession nos. U25789 and L38826) were identified in the search for BRCA1 and predicted to map to chromosome 17q12-21 (Albertsen et al 1994;Harshman et al 1995). U25789 was predicted to map between markers D17S1321 and D17S1325, a -600-kb region containing the BRCA1 gene .…”

“…Within the BRCA1 gene one strong match to the K18 Alu, including two HRE motifs, was found at position 5137 in intron 2. In addition, a recently identified subclass of Alu repeats containing the ERE consensus sequence GGTCAnnnTGGTC(n)9-TGACC can function as estrogen receptordependent transcriptional enhancers in yeast (Norris et al 1995). The BRCA1 contig shows two matches to this consensus sequence in BRCA1 introns 2 and 7 (see also : Norris 1995).…”

“…In addition, a recently identified subclass of Alu repeats containing the ERE consensus sequence GGTCAnnnTGGTC(n)9-TGACC can function as estrogen receptordependent transcriptional enhancers in yeast (Norris et al 1995). The BRCA1 contig shows two matches to this consensus sequence in BRCA1 introns 2 and 7 (see also : Norris 1995). The possibility that Alu repeats containing EREs are functionally significant in regulating BRCA1 expression is particularly appealing, given that BRCA1 is expressed in hormonally responsive tissues (Gudas et al 1995(Gudas et al , 1996Lane et al 1995;Marquis et al 1995;Vaughn et al 1996) and given the epidemiologic association of estrogen and breast cancer risk (Kelsey 1993).…”

Identification and Genetic Mapping of Genes for Hereditary Breast Cancer and Ovarian Cancer in Families Unlinked to BRCA1

“…The most common approaches are (1) exon amplification (Buckler et al, 1991), (2) hybrid selection (Parimoo et al, 1991;Lovett et al, 1991), (3) analysis of genome sequence (UnerBacher and Mural, 1991), and (4) direct cDNA library screening . The advantages and disadvantages of these methods have been described (Hochgeschwender, 1992;Harshman et al, 1995). As it is unlikely that a single method will identify all the transcripts within a given genomic interval (Harshman et al, 1995), we sought to develop a detailed transcription map of the BRCA2 region by employing the first three of these techniques.…”