Figure 2 - uploaded by Erkki Hölttä
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Chromosome walking at the promoter region of the mouse stanniocalcin (stc) gene. The stc bacterial artificial chromosome (BAC) clone DNA was digested with EcoRV, HincII, DraI, ScaI, StuI, and RsaI restriction enzymes and ligated to the L1C vectorette. For chromosome walking, the stc sequences were amplified with a gene-specific primer and the vectorette-specific primer. The map of the 5′ region of stc with the first exon (black box) is shown on the left. The arrow in the map shows the binding site of the gene-specific primer.
Source publication
Chromosome walking in mammalian DNA by vectorette PCR is not always very specific, and the walks have been limited to distances <1 kb. To improve the method, we have designed new vectorettes, which unlike the currently used ones have very little repetitive sequences or homology with known DNA sequences of various origins in the data banks. We have...
Context in source publication
Context 1
... and a 0.9-kb DraI fragment from the 5′ flanking region of the human plat gene ( Figure 1C). Chromosome walk- ing in a mouse stc bacterial artificial chromosome (BAC) clone with stc- specific primers to different restriction enzyme sites in the 5′ region of the stc gene generated fragments ranging from 0.6 to 7 kb in size (Figure 2), which are large fragments even for chromosome walking in cloned DNA. ...
Citations
Transposable element (TE) display is a method exhibiting the insertion site variations of TE, serving for multiple dominant markers. We report an improved TE display method evaluated by testing inheritance pattern of the TE insertion sites in two different piggyBac-like elements in a lepidopteran insect tobacco budworm. The specificity of the polymerase chain reaction (PCR)-based method was confirmed by sequencing randomly chosen a total of 15 bands all revealing the junctions between TE-end and the flanking sequence. The improved method removes the steps in the conventional TE display method using radiolabelled primers and running denaturing gel, saving time and costs.
Germline mutations in the tumor suppressor gene TP53 are associated with high incidence of early-onset malignancies, and somatic mutations occur in 20-40% of all breast cancer cases. We investigated the association of common genetic variation in TP53 and its flanking genes, WDR79 and ATP1B2, with risk for breast cancer. Single nucleotide polymorphisms (SNPs) identified in a re-sequence analysis were genotyped in 2 large case-control studies including 731 cases and 1,124 controls from Norway, and 1,995 cases and 2,296 controls from Poland. Analyses of the pooled data showed no SNPs in TP53 to be significantly associated with risk for breast cancer. However, we found a significant and consistent association with risk for a SNP in exon 1 (R68G) of the 5' neighboring gene WDR79 (rs2287499, OR (95% CI) = 1.08 (0.95-1.23) for CG vs. CC and 1.60 (1.04-2.47) for GG vs. CC, p-trend = 0.01). Stratification by ER and PR status, showed these increases in risk to be limited to ER negative tumors (OR (95% CI) per variant allele: 1.42 (1.18-1.71) p-trend = 0.00009). In addition, 2 TP53 SNPs (rs17887200 3'of STP and rs12951053 in intron 7) showing weak and non-significant overall increases in risk, were also associated with ER negative tumors (1.48 (1.11-1.93) p-trend = 0.01 and 1.29 (1.06-1.58) p-trend = 0.009, respectively). In conclusion, this comprehensive evaluation of common genetic variation in TP53 and its flanking genes found no significant overall associations between SNPs in TP53 and breast cancer risk. However, data suggested that common variation in TP53 or WDR79 could be associated with ER negative breast cancers.
