| 1. |
- Dunham, I, et al.
(författare)
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The DNA sequence of human chromosome 22
- 1999
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Ingår i: Nature. - : Springer Science and Business Media LLC. - 0028-0836 .- 1476-4687. ; 402:6761, s. 489-495
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Tidskriftsartikel (refereegranskat)
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| 6. |
- Pontén, F, et al.
(författare)
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Genomic analysis of single cells from human basal cell cancer using laser-assisted capture microscopy.
- 1997
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Ingår i: Mutation research. - 0027-5107 .- 1873-135X. ; 382:1-2
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Tidskriftsartikel (refereegranskat)abstract
- In this study, we show that direct mutational analysis of genomic DNA can be performed on single somatic cells extracted from a frozen, immunohistochemically stained tissue section using laser-assisted capture microscopy. Eighty-nine single tumor cells were separately dissected from one case of human basal cell cancer (BCC) and p53 mutations were analyzed by direct semi-automated sequencing of PCR fragments. Amplification was obtained for at least one of the two analyzed exons from approximately 50% of the single tumor cells. Identical p53 mutations were found in widely spread areas of the tumor, suggesting a clonal proliferation originating from one cell. Interestingly, comparison between results of immunohistochemistry and genetic analysis of the single cells revealed the same p53 mutations irrespective of the p53 immunoreactivity. We propose that this approach has a great potential to allow investigation of genotypic differences in single cells and more specifically to resolve important and fundamental questions determining cancer heterogeneity.
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| 7. |
- Williams, Cecilia, 1969-, et al.
(författare)
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Assessment of sequence-based p53 gene analysis in human breast cancer : messenger RNA in comparison with genomic DNA targets.
- 1998
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Ingår i: Clinical Chemistry. - : Oxford University Press (OUP). - 0009-9147 .- 1530-8561. ; 44:3
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Tidskriftsartikel (refereegranskat)abstract
- The high prevalence of p53 mutations in human cancers and the suggestion from several groups that the presence or absence of p53 mutations might have both prognostic and therapeutic consequences point to the importance of optimal methods for p53 determination. Several strategies exploring this have been described, based either on mRNA or genomic DNA as a template. However, no comparative study on the reliability of the two templates has been performed. The principal aim of this study was to study the concordance of RNA- and DNA-based direct sequencing methods in detecting p53 mutations in breast tumors. In 100 tumors, 22 mutations were detected by both methods. Furthermore, one stop mutation, two splice-site mutations, and one intron alteration were found only by genomic sequencing. In addition, the comparative study suggests that cells with missense mutations have increased steady-state concentrations of p53-specific mRNA, in contrast to cells with a gene encoding a truncated protein.
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| 10. |
- Odeberg, J, et al.
(författare)
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Context-dependent Taq-polymerase-mediated nucleotide alterations, as revealed by direct sequencing of the ZNF189 gene : implications for mutation detection.
- 1999
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Ingår i: Gene. - 0378-1119 .- 1879-0038. ; 235:1-2
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Tidskriftsartikel (refereegranskat)abstract
- We have recently reported on the genetic organisation of a novel Krüppel-like zinc finger, ZNF189, located to 9q22-q31. In that study we found no mutations in the coding sequence when using ZNF189 as a candidate gene for sporadic basal cell cancer and squamous cell cancer. Here, by direct sequencing of the proximal promotor of ZNF189, mutations were found to appear in a small hot-spot region in over 50% of analysed tumour samples, the majority being G to A substitutions. The hot-spot region spans a 24bp G-rich region. Repeated analyses of the original sample lysates fail to confirm each of these mutations; and frequently new mutations appear at neighbouring positions. Subsequent analysis with serial dilutions of genomic DNA and a cosmid harbouring the wild-type ZNF189 gene demonstrate that these sequence-specific alterations arise in the outer PCR-amplification when 50 copies or less of template are used. Although the mechanism of how these context-specific alterations arise is not proven, the results demonstrate a previously unreported type of PCR-mediated sequence-specific alteration that easily could have been interpreted as being of clinical relevance. The phenomena observed show that mutations detected by direct sequencing can be caused by PCR-introduced alterations. Consequently, this should be of general caution in mutation analysis of disease gene candidates when using small amounts of template, such as microdissected biopsies.
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