Screening Human Genes for Small Alterations Performing an Enzymatic Cleavage Mismatched Analysis (ECMA) Protocol

Vogiatzakis, N.; Kekou, Kyriaki; Sophocleous, Christalena; Kitsiou, Sofia; Mavrou, Ariadni; Bakoula, Chrysa; Kanavakis, Emmanouel

doi:10.1007/s12033-007-0065-6

Cited by 12 publications

(6 citation statements)

References 25 publications

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“…Unnecessary prolonged treatment would increase the background noise with reduced signal-to-noise ratio due to the undesired nonspecific digestions. The recommended incubation time for Surveyor assay is 20-30 min [24,29,30], which is consistent with our observation where the Surveyor is capable to effectively digest mismatches within 30 min ( Fig. 3 and Fig.…”

Section: Discussionsupporting

confidence: 89%

A simple, high sensitivity mutation screening using Ampligase mediated T7 endonuclease I and Surveyor nuclease with microfluidic capillary electrophoresis

Huang

Cheong²,

Lim³

et al. 2012

Electrophoresis

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Mutation and polymorphism detection is of increasing importance for a variety of medical applications, including identification of cancer biomarkers and genotyping for inherited genetic disorders. Among various mutation-screening technologies, enzyme mismatch cleavage (EMC) represents a great potential as an ideal scanning method for its simplicity and high efficiency, where the heteroduplex DNAs are recognized and cleaved into DNA fragments by mismatch-recognizing nucleases. Thereby, the enzymatic cleavage activities of the resolving nucleases play a critical role for the EMC sensitivity. In this study, we utilized the unique features of microfluidic capillary electrophoresis and de novo gene synthesis to explore the enzymatic properties of T7 endonuclease I and Surveyor nuclease for EMC. Homoduplex and HE DNAs with specific mismatches at desired positions were synthesized using PCR (polymerase chain reaction) gene synthesis. The effects of nonspecific cleavage, preference of mismatches, exonuclease activity, incubation time, and DNA loading capability were systematically examined. In addition, the utilization of a thermostable DNA ligase for real-time ligase mediation was investigated. Analysis of the experimental results has led to new insights into the enzymatic cleavage activities of T7 endonuclease I and Surveyor nuclease, and aided in optimizing EMC conditions, which enhance the sensitivity and efficiency in screening of unknown DNA variations.

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Section: Discussionsupporting

confidence: 89%

A simple, high sensitivity mutation screening using Ampligase mediated T7 endonuclease I and Surveyor nuclease with microfluidic capillary electrophoresis

Huang

Cheong²,

Lim³

et al. 2012

Electrophoresis

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“…From the methodological aspect, we would like to comment on the usefulness of the ECMA assay as a screening procedure for the detection of small alterations in the routine molecular diagnosis of DBA. ECMA relies on the ability of the surveyor enzyme to detect and cleave mismatches at both strands thus offering a robust, time saving diagnostic tool towards the detection of point mutations feasible for any molecular laboratory even in developing countries. The high level of consistency between results from ECMA and direct sequencing indicated that ECMA is an inexpensive, simple, and efficient method which in this study allowed screening and disclosure of all alterations reported (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…Primer sequences are available upon request. Prior to direct sequencing, Enzymatic Cleavage Mismatch Analysis (ECMA) was applied as a screening for mutations method. ECMA is based on the formation of heteroduplexes between mutant and wild‐type alleles and the ability of Surveyor TM nuclease to digest both strands of DNA at a mismatch.…”

Section: Methodsmentioning

confidence: 99%

Clinical phenotype and genetic analysis of RPS19, RPL5, and RPL11 genes in Greek patients with Diamond Blackfan Anemia

Delaporta

Sofocleous

Stiakaki

et al. 2014

Pediatric Blood & Cancer

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“…Under specific digestive conditions surveyor nuclease recognizes and cleaves both DNA strands at mismatched nucleotide positions (Surveyor Mutation Detection Kit, No 706020, Transgenomics Inc.) (16). The PCR-generated fragments, including also those without an abnormal ECMA result, were also directly sequenced with the Thermo Sequenase Primer Cycle Sequencing kit (GE Healthcare, UK), according to the manufacturer's instructions, run on Long Read Tower System automated sequencer (VisGen, Ontario, Canada) and analyzed using GeneObject Software (Visible Genetics Inc, Ontario, Canada).…”

Section: Methodsmentioning

confidence: 99%

Phenotypic and Genotypic Variability in Four Males With MECP2 Gene Sequence Aberrations Including a Novel Deletion

et al. 2010

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ABSTRACT:The MECP2 gene mutations cause Rett syndrome (RTT) (OMIM: 312750), an X-linked dominant disorder primarily affecting girls. Until RTT was considered lethal in males, although now approximately 60 cases have been reported. Males with MECP2 mutations present with a broad spectrum of phenotypes ranging from neonatal encephalopathy to nonsyndromic mental retardation (MR). Four boys (aged, 3-11 y) were evaluated for MR. Patient 1 had autistic features. Patients 2 and 3 were brothers both presenting with psychomotor delay. Patient 4 showed dysmorphic features and behavioral problems reminiscent of FXS. All patients had a normal 46, XY karyotype and three were tested for FXS with negative results. MECP2 gene analysis of exons 3 and 4 was performed using methods based on the PCR, including Enzymatic Cleavage Mismatched Analysis (ECMA) and direct sequencing. Patient 1 presented somatic mosaicism for the classic RTT p.R106W mutation and patient 4 carried the p.T203M polymorphism. Analysis of the mothers in both cases revealed normal DNA sequences. Patients 2 and 3 had a novel deletion (c.1140del86) inherited from their unaffected mother. MECP2 gene mutations may be considered a rare cause of MR in males although great phenotypic variation hinders genotype-phenotype correlation. (Pediatr Res 67: 551-556, 2010) R ett syndrome (RTT) (OMIM: 312750) is a severe Xlinked dominant neurodevelopmental disorder, typically affecting females. It is characterized by apparently normal psychomotor development during the first 6 months of life, followed by gradual loss of acquired skills, deceleration of head growth, initiation of stereotypic hand movements, and autistic behavior by the age of 3-4 y (1). During the course of the disease multiple clinical problems emerge such as seizures, scoliosis, breathing abnormalities, and severe mental retardation (MR) (1). The prevalence of RTT is 1:10 -15.000 live births per year and 99% of the cases are considered sporadic (1).Although the diagnosis of classic RTT is based on welldefined criteria, several atypical RTT forms also exist (2). In the majority of cases, the syndrome is caused by heterozygous mutations in the MECP2 gene, which is located on chromosome X (Xq28) and encodes a methyl-CpG binding protein 2 (MeCP2). The protein is thought to selectively bind methyl-CpG islands in the mammalian genome, functioning as a regulator (mostly a repressor) of gene expression. Mutations of the MECP2 gene have been reported in a broad phenotypic spectrum of mentally retarded individuals (3). The first case was molecularly documented in 1999 after the discovery of the MECP2 gene. Generally, males were considered not to survive due to the X-linked mode of inheritance of the disease (4), but to date, more than 60 male patients with MECP2 mutations have been reported (5), suggesting that mutations in this gene may be included among the causes of MR in males. Recent data show that the frequency of MECP2 mutations among mentally retarded males is approximately 1.3 to 1.7% (5).The clinical phenotype...

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Screening Human Genes for Small Alterations Performing an Enzymatic Cleavage Mismatched Analysis (ECMA) Protocol

Cited by 12 publications

References 25 publications

A simple, high sensitivity mutation screening using Ampligase mediated T7 endonuclease I and Surveyor nuclease with microfluidic capillary electrophoresis

A simple, high sensitivity mutation screening using Ampligase mediated T7 endonuclease I and Surveyor nuclease with microfluidic capillary electrophoresis

Clinical phenotype and genetic analysis of RPS19, RPL5, and RPL11 genes in Greek patients with Diamond Blackfan Anemia

Phenotypic and Genotypic Variability in Four Males With MECP2 Gene Sequence Aberrations Including a Novel Deletion

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