Mutation analyses of KRAS exon 1 comparing three different techniques: temporal temperature gradient electrophoresis, constant denaturant capillaryelectrophoresis and allele specific polymerase chain reaction

Bjørheim, Jens; Lystad, Sigrid; Lindblom, Annika; Kressner, Ulf; Westring, Sophia; Wahlberg, Siobhan; Lindmark, Gunilla; Gaudernack, Gustav; Ekstrøm, Per Olaf; Røe, Janne; Thilly, William G.; Børresen-Dale, Anne Lise

doi:10.1016/s0027-5107(98)00057-8

Cited by 55 publications

(59 citation statements)

References 12 publications

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“…Using heteroduplex (hybridized from wild type and mutant homoduplexes) analysis for the detection of single base substitutions leads to a signi®cant increase in detection ef®-ciency (Abrams and Stanton 1992). To date, heteroduplexes have been used for human genetic screening applications using TTGE (Bùrresen-Dale et al 1997;Bjùrheim et al 1998;Marsh et al 1998). To our knowledge, heteroduplex analysis, except for the coanalysis of undesired heteroduplex formation during PCR, has not yet been reported for studies on microbial communities (Muyzer and Smalla 1998).…”

Section: Resultsmentioning

confidence: 99%

“…To calculate the ESEGR for TTGE, the temporal temperature increase (ramping rate) was converted into a hypothetical chemical denaturant increase by using a conversion factor of 0Á3 C temperature increase per 1% chemical denaturant (Abrams and Stanton 1992) and related to the uidA amplicon migration rate in the gel; the amplicon migration rate was determined in a TTGE gel below denaturation conditions for the uidA fragment. The TTGE analysis, performed according to Bùrresen-Dale et al (1997), showed an ESEGR of 2Á3% cm À1 chemical denaturant increase.…”

Section: Estimated Spatial Equivalent Gradient Resolution Of Temporalmentioning

confidence: 99%

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Comparative analysis of denaturing gradient gel electrophoresis and temporal temperature gradient gel electrophoresis in separating Escherichia coli uidA amplicons differing in single base substitutions

Farnleitner

Kreuzinger

Kavka³

et al. 2000

Lett Appl Microbiol

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A set of Escherichia coli freshwater isolates was chosen to compare the effectiveness of denaturing gradient gel electrophoresis (DGGE) vs temporal temperature gradient gel electrophoresis (TTGE) for separating homologous amplicons from the respective uidA region differing in one to seven single base substitutions. Both methods revealed congruent results but DGGE showed a ®ve to eight times higher spatial separation of the uidA amplicons as compared with TTGE, although the experiments were performed at comparable denaturing gradients. In contrast to TTGE, DGGE displayed clear and focused bands. The results strongly indicated a signi®cantly higher discrimination ef®ciency of the spatial chemical denaturing gradient as compared with the temporal temperature denaturing gradient for separating the uidA amplicons. Denaturing gradient gel electrophoresis proved to be highly ef®cient in the differentiation of E. coli uidA sequence types.

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Section: Resultsmentioning

confidence: 99%

Section: Estimated Spatial Equivalent Gradient Resolution Of Temporalmentioning

confidence: 99%

Comparative analysis of denaturing gradient gel electrophoresis and temporal temperature gradient gel electrophoresis in separating Escherichia coli uidA amplicons differing in single base substitutions

Farnleitner

Kreuzinger

Kavka³

et al. 2000

Lett Appl Microbiol

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“…If limit of detection were the main focus then a single capillary system would be the instruments choose. CDCE has a limit of detection down to 10 −3 on a regular basis 9,13,15,36 , but has been used to study mutant fractions down to 10 −6 35, 79 . Furthermore, peak collection and enrichment can be performed on the single capillary system 34-37, 63, 70 .…”

Section: Anticipated Resultsmentioning

confidence: 99%

“…The improved methods have included modes of sequencing DNA [5][6][7] , allele-specific recognition of mutant sequences [8][9][10] and methods that separate point mutant from wild type sequences on the basis of lower thermal stability in wild type/mutant heteroduplexes as opposed to wild type homoduplexes [11][12][13][14][15][16][17][18][19] . A review of all of these contributions is beyond the scope of this effort; here we focus on the technical history and applications of methods that depend on the separation of wild type and mutant sequences on the basis of kinetics of DNA melting and reannealing while DNA is moved by electromotive force through a molecular sieve such as a polyacrylamide gel.…”

Section: Introductionmentioning

confidence: 99%

Analysis of mutational spectra by denaturing capillary electrophoresis

et al. 2008

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Numbers and kinds of point mutant within DNA from cells, tissues and human population may be discovered for nearly any 75-250bp DNA sequence. High fidelity DNA amplification incorporating a thermally stable DNA "clamp" is followed by separation by denaturing capillary electrophoresis (DCE). DCE allows for peak collection and verification sequencing. DCE in a mode of cycling temperature, e.g.+/− 5°C, CyDCE, permits high resolution of mutant sequences using computer defined analytes without preliminary optimization experiments. DNA sequencers have been modified to permit higher throughput CyDCE and a massively parallel,~25,000 capillary system, has been designed for pangenomic scans in large human populations. DCE has been used to define quantitative point mutational spectra for study a wide variety of genetic phenomena: errors of DNA polymerases, mutations induced in human cells by chemicals and irradiation, testing of human genecommon disease associations and the discovery of origins of point mutations in human development and carcinogenesis.

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“…The pellet was incubated with 10 pellet volumes (approximately 500 µl) of lysis buffer (0.32 M sucrose, 10 mM Tris-HCl, 1% (v/v) Triton X-100) and 0.2 volumes of proteinase K (final concentration 400 µg ml -1 ) for 2 to 3 days at 37˚C. DNA was phenol-chloroform extracted and precipitated in ethanol using conventional techniques (Bjorheim et al, 1998). The resulting DNA pellet was resuspended in 50 µl TE buffer, pH 7.4, (10 mM Tris-HCl, pH 7.4, 1 mM EDTA, pH 8.0).…”

Section: K-ras-2 Mutation Analysismentioning

confidence: 99%

Frequent k- ras -2 mutations and p16INK4Amethylation in hepatocellular carcinomas in workers exposed to vinyl chloride

et al. 2001

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Summary Vinyl chloride (VC) is a know animal and human carcinogen associated with liver angiosarcomas (LAS) and hepatocellular carcinomas (HCC). In VC-associated LAS mutations of the K-ras-2 gene have been reported; however, no data about the prevalence of such mutations in VC associated HCCs are available. Recent data indicate K-ras-2 mutations induce P16 methylation accompanied by inactivation of the p16 gene. The presence of K-ras-2 mutations was analysed in tissue from 18 patients with VC associated HCCs. As a control group, 20 patients with hepatocellular carcinoma due to hepatitis B (n = 7), hepatitis C (n = 5) and alcoholic liver cirrhosis (n = 8) was used. The specific mutations were determined by direct sequencing of codon 12 and 13 of the K-ras-2 gene in carcinomatous and adjacent nonneoplastic liver tissue after microdissection. The status of p16 was evaluated by methylation-specific PCR (MSP), microsatellite analysis, DNA sequencing and immunohistochemical staining. All patients had a documented chronic quantitated exposure to VC (average 8883 ppmy, average duration: 245 months). K-ras-2 mutations were found in 6 of 18 (33%) examined VC-associated HCCs and in 3 cases of adjacent non-neoplastic liver tissue. There were 3 G → A point mutations in the tumour tissue. All 3 mutations found in non-neoplastic liver from VC-exposed patients were also G → A point mutations (codon 12-and codon 13-aspartate mutations). Hypermethylation of the 5′ CpG island of the p16 gene was found in 13 of 18 examined carcinomas (72%). Of 6 cancers with K-ras-2 mutations, 5 specimens also showed methylated p16. Within the control group, K-ras-2 mutation were found in 3 of 20 (15%) examined HCC. p16 methylation occurred in 11 out of 20 (55%) patients. K-ras-2 mutations and p16 methylation are frequent events in VC associated HCCs. We observed a K-ras-2 mutation pattern characteristic of chloroethylene oxide, a carcinogenic metabolite of VC. Our results strongly suggest that K-ras-2 mutations play an important role in the pathogenesis of VC-associated HCC. prove the association of their cancer with VC exposure. As a control group, hepatocellular carcinoma tissue from 20 patients with chronic hepatitis B and C virus infection and alcohol-induced cirrhosis with HCC was used. The presence of K-ras-2 mutations was examined by direct sequencing after microdissection of the tumour nodules. MATERIALS AND METHODS Patients and tissue samplesData were extracted from the German industrial professional association for the chemical industry which is responsible for statutory accident insurance and provides medical and social rehabilitation and financial compensation to persons who incur an industrial injury or occupational disease. According to the German social legislation code VII, compensation for work-related impairment and disability is only possible in cases of a proven association of occupational factor and disease. The criteria for the patients included in this study were a documented chronic quantitated exposure to VC, which leads ...

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Mutation analyses of KRAS exon 1 comparing three different techniques: temporal temperature gradient electrophoresis, constant denaturant capillaryelectrophoresis and allele specific polymerase chain reaction

Cited by 55 publications

References 12 publications

Comparative analysis of denaturing gradient gel electrophoresis and temporal temperature gradient gel electrophoresis in separating Escherichia coli uidA amplicons differing in single base substitutions

Comparative analysis of denaturing gradient gel electrophoresis and temporal temperature gradient gel electrophoresis in separating Escherichia coli uidA amplicons differing in single base substitutions

Analysis of mutational spectra by denaturing capillary electrophoresis

Frequent k- ras -2 mutations and p16INK4Amethylation in hepatocellular carcinomas in workers exposed to vinyl chloride

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