Detection of mutations in human genes by a new rapid method: cleavage fragment length polymorphism analysis (CFLPA)

Rossetti, Sandro; Englisch, Sabine; Bresin, Elena; Pignatti, Pier Franco; Turco, Alberto

doi:10.1006/mcpr.1996.0085

Cited by 32 publications

(16 citation statements)

References 14 publications

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“…Further, in some cases it could be difficult to distinguish abnormal bands from normal ones among the many bands expressed in X‐ray films (Figs 1, 3). In this regard, Rossetti et al recently reported that the use of CFLP with silver staining as the detection method resulted in a good outcome 17 …”

Section: Discussionmentioning

confidence: 99%

Mutational analysis of human p53 gene in human gliomas by Cleavase^® fragment length polymorphism

Uehara¹,

Kawano²,

Kataoka

et al. 1999

Neuropathology

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Cleavase® fragment length polymorphism (CFLP) is a newly developed method used for the detection of gene mutation. This study evaluates the usefulness of this method. Thirty‐three human glioma specimens obtained at surgery were analyzed for human p53 gene mutation using radiolabeled polymerase chain reaction (PCR)‐based CFLP analysis and the results were compared with results of direct sequencing. Three (one of five anaplastic astrocytomas and two of 24 glioblastomas) and five (one of four low‐grade gliomas, one of five anaplastic astrocytomas and three of 24 glioblastomas) samples were suspected as being mutations in exon 5–6 and 7–8 of the p53 gene, respectively. Further, two of three (one anaplastic astrocytoma and one glioblastoma) and two of five (both glioblastoma) samples were confirmed as having mutations in direct sequence (their positive rates were 66.7 and 40%, respectively). However, the false‐negative rate of CFLP analysis was very low (0%, 0/60). Therefore, it was concluded that although PCR‐CFLP using a radioisotope might be too sensitive given its superior false‐negative rate, it offers an alternative protocol for the screening of the mutation of human p53 gene.

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Section: Discussionmentioning

confidence: 99%

Mutational analysis of human p53 gene in human gliomas by Cleavase^® fragment length polymorphism

Uehara¹,

Kawano²,

Kataoka

et al. 1999

Neuropathology

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“…CFLP also has more advantages than SSCP; for example, the pattern of objective electrophoresis roughly defines a mutative position of objective DNA. [14][15][16][17][18]…”

Section: Cleavase ® Fragment Length Polymorphism Versus Single-strandmentioning

confidence: 99%

“…It also has more advantages such as the fact that the pattern of objective electrophoresis roughly defines a mutative position of a subjective DNA. [14][15][16][17][18] Therefore, in an attempt to analyze genetic mutations present in human glioma cells, we have employed the CFLP method. The principal aim of this study was to evaluate the efficiency and accuracy of CFLP for mutation detection of human glioma tissues.…”

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confidence: 99%

Mutational analysis of human p53 gene in human gliomas by CleavaseR fragment length polymorphism

Uehara¹,

Kawano²,

Kataoka³

et al. 1999

Neuropathology

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human p53, 7 EGFR, 8 FGFR2, 9 MDM2, 10 NF2, 11 p16 12 and PTEN/MMAC1 13 genes were reported.For genetic mutation detection, single-strand conformation polymorphism (SSCP) has been widely used recently but requires some special equipment and strict maintenance of reactive temperature. Cleavase ® fragment length polymorphism (CFLP) was developed recently to eliminate the disadvantages of SSCP. The procedure is able to be finished within 1 day and does not require special equipment. It also has more advantages such as the fact that the pattern of objective electrophoresis roughly defines a mutative position of a subjective DNA. [14][15][16][17][18] Therefore, in an attempt to analyze genetic mutations present in human glioma cells, we have employed the CFLP method. The principal aim of this study was to evaluate the efficiency and accuracy of CFLP for mutation detection of human glioma tissues. We screened the p53 gene mutation by CFLP and compared this with results from direct sequence analysis.

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“…Because single nucleotide changes affect the stability of localized secondary structures, the resulting CFLP fingerprints generally contain pattern changes in the region of a given mutation [10]. Therefore, samples which contain single-base pair changes within common nucleic acid sequences will retain the same structural fingerprint for those areas of shared sequence while demonstrating pattern changes in areas of sequence divergence [11,12]. These pattern changes can be identified by the appearance or disappearance of one or more bands, as well as by intensity changes in existing bands.…”

Section: Introductionmentioning

confidence: 99%

Detection ofp53 gene mutation: Analysis by single-strand conformation polymorphism and Cleavase fragment length polymorphism

et al. 1999

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We have generated a collection of clones containing single point mutations within the exon 5—9 hot spot regions of the p53 gene by using polymerase chain reaction (PCR) to amplify select regions of the gene from characterized cell lines. These clones were then used to address the sensitivity of mutation detection using slab‐gel single‐strand conformation polymorphism (SSCP) and Cleavase fragment length polymorphism (CFLP) assay systems. Both methods exhibited high sensitivities for the detection of mutations in cloned p53 mutations in this study: 97% for CFLP and 94% for SSCP. In addition to resulting in higher sensitivity of mutation detection, CFLP has the capability to analyze longer fragments. In this study, CFLP identified five intronic mutations which were not investigated in the exon‐specific SSCP assay. These results agree with those found elsewhere and demonstrate that CFLP scanning can have practical advantages when used for the identification of sequence alterations within the p53 gene.

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Detection of mutations in human genes by a new rapid method: cleavage fragment length polymorphism analysis (CFLPA)

Cited by 32 publications

References 14 publications

Mutational analysis of human p53 gene in human gliomas by Cleavase^® fragment length polymorphism

Mutational analysis of human p53 gene in human gliomas by Cleavase^® fragment length polymorphism

Mutational analysis of human p53 gene in human gliomas by CleavaseR fragment length polymorphism

Detection ofp53 gene mutation: Analysis by single-strand conformation polymorphism and Cleavase fragment length polymorphism

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Detection of mutations in human genes by a new rapid method: cleavage fragment length polymorphism analysis (CFLPA)

Cited by 32 publications

References 14 publications

Mutational analysis of human p53 gene in human gliomas by Cleavase® fragment length polymorphism

Mutational analysis of human p53 gene in human gliomas by Cleavase® fragment length polymorphism

Mutational analysis of human p53 gene in human gliomas by CleavaseR fragment length polymorphism

Detection ofp53 gene mutation: Analysis by single-strand conformation polymorphism and Cleavase fragment length polymorphism

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Mutational analysis of human p53 gene in human gliomas by Cleavase^® fragment length polymorphism

Mutational analysis of human p53 gene in human gliomas by Cleavase^® fragment length polymorphism