Detection of Virtually All Mutations-SSCP (DOVAM-S): A Rapid Method for Mutation Scanning with Virtually 100% Sensitivity

Liu, Q.; Feng, Jinong; Buzin, Carolyn H.; Wen, C.; Nozari, G.; Mengos, April; Nguyen, Vu Quoc Huy; Liu, J.; Crawford, L. V.; Fujimura, Frank K.; Sommer, Steve S.

doi:10.2144/99265rr03

Cited by 72 publications

(66 citation statements)

References 13 publications

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“…more PCR primers and more PCR reactions are required. To overcome the size limitation, a new method combining dideoxyfingerprinting and SSCP, named "detection of virtually all mutation-SSCP" (DOVAM-S), was developed [79][80][81]. However, DOVAM-S requires several gel running conditions to attain detection of virtually all mutations, making it relatively inconvenient as well.…”

Section: Discussionmentioning

confidence: 99%

Development of a simple and highly sensitive mutation screening system by enzyme mismatch cleavage with optimized conditions for standard laboratories

Tsuji

Niida

2008

Electrophoresis

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Efficient screening of unknown DNA variations is one of the substantive matters of molecular biology even today. Historically, single-strand conformation polymorphism (SSCP) and heteroduplex analysis (HA) are the most commonly used methods for detecting DNA variations everywhere in the world because of their simplicity. However, the sensitivity of these methods is not satisfactory for screening purpose. Recently, several new PCR based mutation screening methods have been developed, but most of them require special instruments and adjustment of conditions for each DNA sequence to attain the maximum sensitivity, eventually becoming as inconvenient as old methods. Enzyme mismatch cleavage (EMC) is potentially an ideal screening method. With high performance nucleases and once experimental conditions are optimized, it requires only conventional staff and conditions remain the same for each PCR product. In this study we tested four commercially available endonucleases for EMC and optimized the electrophoresis and developing conditions. We prepared 25 known DNA variations consisting of 18 single base substitutions (8 transitions and 10 transversions, including all possible sets of mismatches) and 7 small deletions or insertions. The combination of CEL nuclease, 12% polyacrylamide gel electrophoresis and rapid silver staining can detect all types of mutations and achieved 100% sensitivity.

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Section: Discussionmentioning

confidence: 99%

Development of a simple and highly sensitive mutation screening system by enzyme mismatch cleavage with optimized conditions for standard laboratories

Tsuji

Niida

2008

Electrophoresis

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“…DOVAM-S is a robotically enhanced, highly redundant modification of SSCP that detected 240 of 240 sequence changes in three blinded analyses (180 different mutations) (Liu et al, 1999;Buzin et al, 2000). Genomic DNA at a concentration of 40 ng/l were used to generate 61 separate PCR segments labeled with 33 P, which included all coding exons of FMO1-6, including the splice junctions.…”

Section: Methodsmentioning

confidence: 99%

“…Such knowledge may be important in future efforts to predict the response to pharmaceuticals and environmental toxicants. Mutation scanning using a robotically enhanced scanning method that detects virtually all mutations (Liu et al, 1999;Buzin et al, 2000) has been employed to detect sequence variation in the complete coding and splicing-regions of the six genes in the human FMO gene family.…”

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confidence: 99%

Identification of Novel Variants of the Flavin-Containing Monooxygenase Gene Family in African Americans

Furnes¹,

Feng²,

Sommer³

et al. 2003

Drug Metab Dispos

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ABSTRACT:Sequence polymorphisms in enzymes involved in drug metabolism have been widely implicated in the differences observed in the sensitivity to various xenobiotics. The flavin-containing monooxygenase (FMO) gene family in humans catalyzes the monooxygenation of numerous N-, P-and S-containing drugs, pesticides, and environmental toxicants. Six genes (FMO1-6) have been identified so far, but the major alleles of FMO2 and FMO6 encode nonfunctional proteins due to a nonsense mutation and splice-site abnormalities, respectively. Data on structural variants exist for human FMO2 and 3, whereas very little is known about the other FMO genes. FMO1-6 were scanned in 50 individuals of African-American descent using the method, detection of virtually all mutationssingle-strand conformational polymorphism. A total of 49 sequence variants were identified in a total 1.35 megabases of scanned sequence, of which 29 were variants affecting protein structure or expression. Some of these are expected to affect the activity of the protein, including a nonsense mutation in FMO1 (R502X) and missense mutations in FMO1 (I303T), FMO4 (E339Q), and FMO5 (P457L) that occur in highly conserved amino acids. Additional deleterious substitutions in FMO2 (del337G) and FMO6 (Q105X) were also identified. Multiple structural variants in the FMO gene family were observed in this African-American sample. Some of the substitutions identified in this study might be useful markers in future association studies assessing sensitivity to environmental toxicants and common disease.

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“…The coding region of MECP2 comprising exons 2-4, exonintron boundaries, and its whole 3 0 UTR, were scanned for mutations with Detection Of Virtually All Mutations-SSCP (DOVAM-S) [Liu et al, 1999]. DOVAM-S is a robotically enhanced and highly redundant form of Single Strand Conformational Polymorphism (SSCP) with virtually 100% sensitivity of mutation detection.…”

Section: Mecp2 Mutation Detectionmentioning

confidence: 99%

MECP2 coding sequence and 3′UTR variation in 172 unrelated autistic patients

Coutinho

Oliveira²,

Katz

et al. 2007

American J of Med Genetics Pt B

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Mutations in the coding sequence of the methyl‐CpG‐binding protein 2 gene (MECP2), which cause Rett syndrome (RTT), have been found in male and female autistic subjects without, however, a causal relation having unequivocally been established. In this study, the MECP2 gene was scanned in a Portuguese autistic population, hypothesizing that the phenotypic spectrum of mutations extends beyond the traditional diagnosis of RTT and X‐linked mental retardation, leading to a non‐lethal phenotype in male autistic patients. The coding region, exon–intron boundaries, and the whole 3′UTR were scanned in 172 patients and 143 controls, by Detection of Virtually All Mutations‐SSCP (DOVAM‐S). Exon 1 was sequenced in 103 patients. We report 15 novel variants, not found in controls: one missense, two intronic, and 12 in the 3′UTR (seven in conserved nucleotides). The novel missense change, c.617G > C (p.G206A), was present in one autistic male with severe mental retardation and absence of language, and segregates in his maternal family. This change is located in a highly conserved residue within a region involved in an alternative transcriptional repression pathway, and likely alters the secondary structure of the MeCP2 protein. It is therefore plausible that it leads to a functional modification of MeCP2. MECP2 mRNA levels measured in four patients with 3′UTR conserved changes were below the control range, suggesting an alteration in the stability of the transcripts. Our results suggest that MECP2 can play a role in autism etiology, although very rarely, supporting the notion that MECP2 mutations underlie several neurodevelopmental disorders. © 2007 Wiley‐Liss, Inc.

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Detection of Virtually All Mutations-SSCP (DOVAM-S): A Rapid Method for Mutation Scanning with Virtually 100% Sensitivity

Cited by 72 publications

References 13 publications

Development of a simple and highly sensitive mutation screening system by enzyme mismatch cleavage with optimized conditions for standard laboratories

Development of a simple and highly sensitive mutation screening system by enzyme mismatch cleavage with optimized conditions for standard laboratories

Identification of Novel Variants of the Flavin-Containing Monooxygenase Gene Family in African Americans

MECP2 coding sequence and 3′UTR variation in 172 unrelated autistic patients

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