Double-Gradient DGGE for Optimized Detection of DNA Point Mutations

Cremonesi, Laura; Firpo, S; Ferrari, Maurizio; Righetti, P. G.; Gelfi, Cecilia

doi:10.2144/97222rr01

Cited by 104 publications

(69 citation statements)

References 16 publications

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“…14,15 In brief, the PCR products were loaded on a 10-70% denaturant gradient gel together with a fully methylated control (In vitro methylated DNA) and an unmethylated control (peripheral blood lymphocytes). After electrophoresis, gels were stained in Tris/EDTA buffer containing ethidium bromide and photographed under ultraviolet transillumination.…”

Section: Methylation Analysis By Dggementioning

confidence: 99%

“…Samples were scored as methylated when bands or smears were present on the gels in the area below the band corresponding to unmethylated DNA as reported in previous publications. 14,15 Bisulfite pyrosequencing Pyrosequencing of the p15 promoter region was performed using the PSQHS96 system (Biotage AB, Uppsala, Sweden) including PyroGold reagents as previously described. 16 Commercially …”

Section: Methylation Analysis By Dggementioning

confidence: 99%

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Gene-specific and global methylation patterns predict outcome in patients with acute myeloid leukemia

et al. 2010

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This study was designed to analyze the effect of global and gene-specific DNA methylation patterns on the outcome of patients with acute myeloid leukemia (AML). Methylation of CDKN2B (p15), E-cadherin (CDH) and hypermethylated in cancer 1 (HIC1) promoters and global DNA methylation by luminometric methylation assay (LUMA) was analyzed in 107 AML patients and cytogenetic and molecular mutational analysis was performed. In addition, genome-wide promoterassociated methylation was assessed using the Illumina HumanMethylation27 array in a proportion of the patients. Promoter methylation was discovered in 66, 66 and 51% of the patients for p15, CDH and HIC1, respectively. In multivariate analysis, low global DNA methylation was associated with higher complete remission rate (hazard ratio (HR) 5.9, P ¼ 0.005) and p15 methylation was associated with better overall (HR 0.4, P ¼ 0.001) and disease-free survival (HR 0.4, P ¼ 0.016). CDH and HIC1 methylation were not associated with clinical outcome. Mutational status and karyotype were not significantly associated with gene-specific methylation or global methylation. Increased genome-wide promoter-associated methylation was associated with better overall and disease-free survival as well as with LUMA hypomethylation. We conclude that global and gene-specific methylation patterns are independently associated with the clinical outcome in AML patients.

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Section: Methylation Analysis By Dggementioning

confidence: 99%

Section: Methylation Analysis By Dggementioning

confidence: 99%

Gene-specific and global methylation patterns predict outcome in patients with acute myeloid leukemia

et al. 2010

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“…After electrophoresis, the gels were stained in Tris acetate/EDTA buffer containing ethidium bromide (2 mg/ml) and photographed under UV transillumination. Samples were scored as methylated when bands were present on the gels below the band corresponding to the unmethylated control (Cremonesi et al, 1997;Aggerholm et al, 2006).…”

Section: Polymerase Chain Reaction (Pcr) and Denaturing Gradient Gel mentioning

confidence: 99%

Maintenance treatment with azacytidine for patients with high‐risk myelodysplastic syndromes (MDS) or acute myeloid leukaemia following MDS in complete remission after induction chemotherapy

et al. 2010

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SummaryThis prospective Phase II study is the first to assess the feasibility and efficacy of maintenance 5-azacytidine for older patients with high-risk myelodysplastic syndrome (MDS), chronic myelomonocytic leukaemia and MDS-acute myeloid leukaemia syndromes in complete remission (CR) after induction chemotherapy. Sixty patients were enrolled and treated by standard induction chemotherapy. Patients that reached CR started maintenance therapy with subcutaneous azacytidine, 5/28 d until relapse. Promoter-methylation status of CDKN2B (P15 ink4b), CDH1 and HIC1 was examined pre-induction, in CR and 6, 12 and 24 months post CR. Twentyfour (40%) patients achieved CR after induction chemotherapy and 23 started maintenance treatment with azacytidine. Median CR duration was 13AE5 months, >24 months in 17% of the patients, and 18-30AE5 months in the four patients with trisomy 8. CR duration was not associated with CDKN2B methylation status or karyotype. Median overall survival was 20 months. Hypermethylation of CDH1 was significantly associated with low CR rate, early relapse, and short overall survival (P = 0AE003). 5-azacytidine treatment, at a dose of 60 mg/m 2 was well tolerated. Grade III-IV thrombocytopenia and neutropenia occurred after 9AE5 and 30% of the cycles, respectively, while haemoglobin levels increased during treatment. 5-azacytidine treatment is safe, feasible and may be of benefit in a subset of patients.

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“…Others, such as denaturing HPLC (13,14 ) can identify unknown single-base mutations in a given sequence. We describe a mutation screening method based on double-gradientdenaturing-gradient gel electrophoresis (DG-DGGE) (15,16 ), which relies on wild-type (WT)/mutant heteroduplex formation. The entire BCR-ABL TK domain covers 4 exons of the ABL gene; therefore, the analysis was performed at the RNA level by means of 2 overlapping PCR fragments (5Ј and 3Ј in Fig.…”

Section: Double-gradient-denaturing-gradient Gel Electrophoresis For mentioning

confidence: 99%

Double-Gradient–Denaturing-Gradient Gel Electrophoresis for Mutation Screening of the BCR-ABL Tyrosine Kinase Domain in Chronic Myeloid Leukemia Patients

Sorel

Chazelas

Brizard³

et al. 2005

Clinical Chemistry

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Double-Gradient DGGE for Optimized Detection of DNA Point Mutations

Abstract: ABSTRACT

Cited by 104 publications

References 16 publications

Gene-specific and global methylation patterns predict outcome in patients with acute myeloid leukemia

Gene-specific and global methylation patterns predict outcome in patients with acute myeloid leukemia

Maintenance treatment with azacytidine for patients with high‐risk myelodysplastic syndromes (MDS) or acute myeloid leukaemia following MDS in complete remission after induction chemotherapy

Double-Gradient–Denaturing-Gradient Gel Electrophoresis for Mutation Screening of the BCR-ABL Tyrosine Kinase Domain in Chronic Myeloid Leukemia Patients

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