Constant denaturant gel electrophoresis, a modification of denaturing gradient gel electrophoresis, in mutation detection

Hovig, Eivind; Smith‐Sørensen, Birgitte; Brøgger, Anton; Børresen, Anne Lise

doi:10.1016/0165-7992(91)90108-g

Cited by 123 publications

(50 citation statements)

References 6 publications

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“…All 16 exons of hMSH2 were screened for mutations using constant dénaturant gel electrophoresis (CDGE) [33]. The separation principle of CDG E is based on the unique melting behaviour of each D N A fragment.…”

Section: Hm Sh2 Mutation Analysismentioning

confidence: 99%

Benign and malignant thyroid lesions show instability at microsatellite loci

Soares

Santos

Seruca

et al. 1997

European Journal of Cancer

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Forty-six benign and m alignant tum ours and tu m ou r-lik e lesions o f the thyroid were analysed for m icrosatellite instability (MI) at eight loci, m ap p in g to four different ch rom osom es, 7 lesio n s (15%) displayed MI at one or more loci, in clu d in g 2/13 nodular goitres, 2/15 follicular ad en om as, 2/12 papillary carcinom as and 1/4 follicular carcin o m as. Two benign and one m alignant lesio n am ong the seven unstable cases exhibited this p h en otyp e at three or m ore loci. We found no m u tation s in the m ism a tch repair gene, hMSH2, in the seven affected cases, after screening all th e exons by CDGE m utation analysis. At variance w ith the data on record, these results indicate that, despite being relatively infrequent, MI does occur n ot only in thyroid carcinom as but also in benign lesion s (goitres and follicular adenom as of the thyroid).

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Section: Hm Sh2 Mutation Analysismentioning

confidence: 99%

Benign and malignant thyroid lesions show instability at microsatellite loci

Soares

Santos

Seruca

et al. 1997

European Journal of Cancer

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“…1). Previous studies (19,21,23) have shown that using a GC clamp increases the number of detectable mutations from 40%o of all single base changes to close to 100%, if they reside in the lower-melting domains of the fragment. Most of the mutations in these codons (Fig.…”

mentioning

confidence: 99%

“…To develop a screening method for p53 mutations, we have tested a modification of the DGGE system (18), termed constant denaturant gel electrophoresis (CDGE) (21 (22). Thirtytwo of the samples that had LOH for at least one of a panel of 17p markers were used for CDGE analysis.…”

mentioning

confidence: 99%

Constant denaturant gel electrophoresis as a rapid screening technique for p53 mutations.

Børresen

Hovig²,

Smith‐Sørensen

et al. 1991

Proc. Natl. Acad. Sci. U.S.A.

162

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At present, mutation of the p53 gene appears to be the most common genetic alteration found in human cancers. These mutations can occur within many different regions of the gene. We have developed a modification of denaturing gradient gel electrophoresis termed "constant denaturant gel electrophoresis" (CDGE), which provides a rapid and sensitive method to screen the four conserved regions within the p53 gene where the majority of p53 mutations have been reported. The sensitivity of CDGE was first tested with known p53 mutations in all four conserved regions. The CDGE technique was then used to screen 32 breast carcinomas that had been analyzed by immunohistochemical methods for altered p53 protein levels and whose DNA had already been shown to have loss of heterozygosity for a chromosome 17p marker. By immunostalning techniques, only 6 of the 32 tumors had elevated p53 expression. However, CDGE detected p53 mutations in 11 of the 32 tumors. DNA sequence analysis was performed to determine the nucleotide positions of these mutations in all 11 samples. Loss of heterozygosity for the pYNZ22 or pl44D6 markers did not associate with either the loss of heterozygosity at the p53 locus or the mutations detected by CDGE. We conclude that CDGE is a rapid and effective technique to screen for p53 mutations.A wide variety of human tumors have been shown to be associated with changes in the p53 gene (1-8). As the list of cancers with p53 mutations increases, there is a need for an accurate and rapid screening technique for these mutations. Unlike some of the dominant oncogenes, p53 can be inactivated by a diverse set of mutations scattered through several important regions of the gene (7). Many recent surveys of p53 mutations have used loss of heterozygosity (LOH) at polymorphic markers closely linked to p53 as an indirect assay for p53 inactivation (9-11). While such a screen is rapid, it cannot detect point mutations, and its accuracy is limited, since some of the nearby polymorphic markers might undergo deletions that do not extend into the p53 locus. Alternatively, because mutations in the p53 gene frequently result in a mutant p53 protein that is significantly overexpressed compared with the low levels of the wild-type protein, other p53 surveys have used immunostaining techniques to screen for mutations (12)(13)(14). Unfortunately, the p53 mutations that result in either a lack of protein or the same low levels of p53 as are present in cells containing the wild-type p53 cannot be detected by this method. The most sensitive screening technique is to sequence the genomic region encoding p53. Although sequencing is quite sensitive, it is also laborintensive. There are several new nucleic acid-based screening methods that can rapidly detect mutations within short fragments of DNA. These techniques include RNase protection assays (15), single-strand conformational polymorphisms (SSCP) (16, 17), denaturing gradient gel electrophoresis (DGGE) (18,19), and detection of base-pair mismatches with hydroxylamine and osmiu...

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“…The four amplified products from each tumour were screened for TP53 mutations using constant denaturant gel eectrophoresis (CDGE) (Borresen et al, 1991;Hovig et al, 1991 The gels, which had the same chemicals and ekctrophoresis conditions as the constant denaturing gels, were run for 2 h with the gradient spanning from 10% to 70% denaturant. Gradient gels were stained and photographed usng both ethidium bromide, as described above, and SYBR green I (Molkuar Probes, Eugene, OR, USA) diluted 1:10000.…”

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confidence: 99%

TP53 gene mutations and protein accumulation in primary vaginal carcinomas

Skomedal

Kristensen²,

Helland³

et al. 1995

Br J Cancer

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Summary Primary carcinomas from 46 patients were screened for TP53 alterations. Immunohistochemistry demonstrated nuclear TPS3 protein accumulation in 22 (48%) cases using the polyclonal CM1 antiserum, whereas 15 (33%) cases showed positive nuclear staining with the mononuclear antibody PAb 1801. Constant denaturant gel electrophoresis (CDGE) was used to screen 27 of the vaginal carcinomas for mutations in the conserved regions of the TP53 gene (exons 5-8 (Lane and Benchimol, 1990). Although the precise mechanism by which the TP53 protein participates in these cellular functions is not fully understood, several biochemical features of TP53 have been elucidated. The TP53 protein is able to regulate transcription directly (Kern et al., 1991;El-Deiry et al., 1992) or by interacting with other transcriptional regulatory factors, such as the TATA-and CAAT-binding proteins (Seto et al., 1992;Agoff et al., 1993). The TP53 protein has also been shown to act as a specific transcription factor controlling the expression of growth arrest genes such as GADD45 (Kastan et al., 1992) and WAF-I (El-Deiry et al., 1993).Mutations in the TP53 gene are the most frequent genetic alteration found in human tumours (Hollstein et al., 1994; Levine et al., 1994

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Constant denaturant gel electrophoresis, a modification of denaturing gradient gel electrophoresis, in mutation detection

Cited by 123 publications

References 6 publications

Benign and malignant thyroid lesions show instability at microsatellite loci

Benign and malignant thyroid lesions show instability at microsatellite loci

Constant denaturant gel electrophoresis as a rapid screening technique for p53 mutations.

TP53 gene mutations and protein accumulation in primary vaginal carcinomas

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