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Results for “mutation scanning”

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At least 73 records · Page 4Linked to original sources
PubMed2001

Mutational scanning of the ABCR gene with double-gradient denaturing-gradient gel electrophoresis (DG-DGGE) in Italian Stargardt disease patients.

Mutations in the retina-specific ABC transporter (ABCR) gene are responsible for autosomal recessive Stargardt disease (arSTGD). Mutation detection efficiency in ABCR in arSTGD patients ranges between 30% and 66% in previously published studies, because of high allelic heterogeneity and technical limitations of the employed methods. Conditions were developed to screen the ABCR gene by double-gradient denaturing-gradient gel electrophoresis. The efficacy of this method was evaluated by analysis of DNA samples with previously characterized ABCR mutations. This approach was applied to mutation detection in 44 Italian arSTGD patients corresponding to 36 independent genomes, in order to assess the nature and frequency of the ABCR mutations in this ethnic group. In 34 of 36 (94.4%) STGD patients, 37 sequence changes were identified, including 26 missense, six frameshift, three splicing, and two nonsense variations. Among these, 20 had not been previously described. Several polymorphisms were detected in affected individuals and in matched controls. Our findings extend the spectrum of mutations identified in STGD patients and suggest the existence of a subset of molecular defects specific to the Italian population. The identification of at least two disease-associated mutations in four healthy control individuals indicates a higher than expected carrier frequency of variant ABCR alleles in the general population. Genotype-phenotype analysis in our series showed a possible correlation between the nature and location of some mutations and specific ophthalmoscopic features of STGD disease.

ATP-Binding Cassette Transporters↗
PubMed2003

Rapid denaturing high-performance liquid chromatography (DHPLC) for mutation scanning of the transforming growth factor beta3 gene using a novel proof-reading polymerase.

We have utilized a novel variation on the conventional denaturing high-performance liquid chromatography (DHPLC) technology, which we term rapid DHPLC, combining changes in instrumentation, cartridge technology and analysis conditions to enable significant increases in throughput to be achieved. In addition, the use of a novel proof-reading polymerase for sample amplification with a low misincorporation rate enables simplification of the DHPLC patterns and hence enhanced mutation detection recognition. This scheme for increasing DHPLC throughput has been tested by scanning the transforming growth factor (TGF) beta3 gene for the presence of mutations for which there is limited published or on-line data available regarding the presence of gene polymorphisms. TGFbeta isoforms have multiple roles in cell division, growth, proliferation, transformation and differentiation. TGFbeta3 is a TGFbeta cytokine isoform, and has an important role in embryogenesis, cell differentiation and wound healing. The TGFbeta3 gene consists of seven exons and six introns spanning 43 000 bp of the human genome on chromosome 14q23-24. The rapid DHPLC approach enabled scanning of all seven exons and part of the promoter region (1000 bp upstream from exon 1 in the 5'-flanking regions) of the TGFbeta3 gene in 95 Caucasian individuals in only 8 days, in comparison to the 17 days it would have previously taken. Mutations were clearly identified in the promoter region of the TGFbeta3 gene but were absent from the exonic regions. Understanding the genetic variations affecting the TGFbeta3 gene is important as this molecule has multiple regulatory functions on a variety of cell types.

Adult↗
PubMed2002

Pyrophosphorolysis-activatable oligonucleotides may facilitate detection of rare alleles, mutation scanning and analysis of chromatin structures.

Pyrophosphorolysis-activated polymerization (PAP) was initially developed to enhance the specificity of allele-specific PCR for detection of known mutations in the presence of a great excess of wild-type allele. The high specificity of PAP derives from the serial coupling of pyrophosphorolysis-mediated activation of a pyrophosphorolysis-activatable oligonucleotide (P*) followed by extension of the activated oligonucleotide. Herein, we demonstrate that genetically engineered DNA polymerases greatly improve the efficiency of PAP, making it a practical technique for detection of rare mutations. We also show that P* oligonucleotides have the novel and unexpected property of high sensitivity to mismatches throughout at least the 16 3'-terminal nucleotides. Thus, PAP constitutes a technology platform of potential utility whenever high specificity is required along the length of an oligonucleotide.

Alleles↗
PubMed2002

Rapid parallel mutation scanning of gene fragments using a microelectronic protein-DNA chip format.

We have developed a method for the de novo discovery of genetic variations, including single nucleotide polymorphisms and mutations, on microelectronic chip devices. The method combines the features of electronically controlled DNA hybridisation on open-format microarrays, with mutation detection by a fluorescence-labelled mismatch- binding protein. Electronic addressing of DNA strands to distinct test sites of the chip allows parallel analysis of several individuals, as demonstrated for mutations in different exons of the p53 gene. This microelectronic chip-based mutation discovery assay may substitute for time-consuming sequencing studies and will complement existing technologies in genomic research.

Adenosine Triphosphatases↗
PubMed1999

Use of DNA toolbox for the characterization of mutation scanning methods. II: evaluation of single-strand conformation polymorphism analysis.

Single-strand conformation polymorphism (SSCP) is one of the most commonly used methods for searching for unknown base changes (mutations). In order to characterize systematically the effects of important physical parameters on the sensitivity and specificity of SSCP, we used the DNA toolbox constructed as described in the companion paper [2]. Using this set of DNA molecules as polymerase chain reaction (PCR) templates, amplicons of various lengths with the same base, mutated to all other bases, were generated. The behavior of these constructs in manual and automated SSCP was analyzed as a function of the size, overall base content of the fragment, nature and location of the base change, and the temperature and pH of electrophoresis. Our results demonstrate that all of these variables interact to determine the rate of detection of single-base changes, with the GC content being the predominant determinant of detection sensitivity.

DNA↗
PubMed1999

Use of a DNA toolbox for the characterization of mutation scanning methods. I: construction of the toolbox and evaluation of heteroduplex analysis.

A systematic characterization of the effects of important physical parameters on the sensitivity and specificity of methods in searching for unknown base changes (mutations or single nucleotide polymorphisms) over a relatively long DNA segment has not been previously reported. To this end, we have constructed a set of molecules of varying G+C content (40, 50, and 60% GC) having all possible base changes at a particular location - the "DNA toolbox". Exhaustive confirmatory sequencing demonstrated that there were no other base changes in any of the clones. Using this set of clones as polymerase chain reaction (PCR) templates, amplicons of various lengths with the same base mutated to all other bases were generated. The behavior of these constructs in manual and automated heteroduplex analysis was analyzed as a function of the size and overall base content of the fragment, the nature and location of the base change. Our results show that in heteroduplex analysis, the nature of the mismatched base pair is the overriding determinant for the ability to detect the mutation, regardless of fragment length, GC content, or the location of the mutation.

DNA, Viral↗
PubMed1996

Mutational scanning of large genes by extensive PCR multiplexing and two-dimensional electrophoresis: application to the RB1 gene.

With the rapid increase in the number of identified human disease genes, the development of accurate and cost-efficient mutation tests has become opportune. Here we present a combination of extensive PCR multiplexing and two-dimensional (2-D) DNA electrophoresis to screen for mutations in 26 exons of the retinoblastoma (RB1) tumor suppressor gene. In 2-D electrophoresis, fragments are separated according to size and base pair sequence in non-denaturing and denaturing gradient gels, respectively. All target fragments, designed to have optimal melting characteristics, were prepared in a two-step PCR (a 6-plex long-PCR pre-amplification and a subsequent 25-plex short-PCR) followed by heteroduplexing. The mixture of PCR amplicons was then subjected to 2-D electrophoresis under a single set of experimental conditions. With this design, 35 previously identified mutations in 18 different exons were detected in 33 bilateral retinoblastoma patients. These results suggest that 2-D electrophoresis in this format provides a generally applicable, practical and fast way to diagnose with high accuracy large genes for a broad spectrum of possible disease-causing mutations.

DNA Mutational Analysis↗
PubMed1996

Extensive mutation scanning of RET in sporadic medullary thyroid carcinoma and of RET and VHL in sporadic pheochromocytoma reveals involvement of these genes in only a minority of cases.

Sporadic medullary thyroid carcinoma (MTC) and pheochromocytoma (PC) have been reported to be associated with some specific RET gene mutations. To assess the role of RET in the development of MTC and PC, we screened 14 sporadic MTC, two MTC-derived cell lines, and 5 sporatic PC cases of RET mutations by a systematic analysis of the whole coding sequence, including all intron-exon junctions. In only 6 of the 14 sporadic MTC we were able to detect a RET mutation. Apart from the MET918-->Thr mutation in 5 of the MTC cases, we found a 3-bp deletion in exon 11, only present in the tumor, in another case. Analysis of 2 cell lines revealed the Met918-->Thr mutation in 1 and a Cys634-->Trp mutation in the other cell line. A possible somatic nature of these mutations could not be confirmed because in neither case was constitutive DNA available. We conclude that a large proportion of sporadic MTC must be due to mutations in an unidentified gene(s) other than RET. In none of the sporadic PC cases was a RET mutation found. As PC is a frequent complication in families suffering from von Hippel Lindau disease, for which mutations of the VHL gene are responsible, we also screened the 5 sporadic PC cases for VHL mutations. This revealed a Gly164-->Ser mutation in a single specimen. Thus, in PC, a large majority of tumors are due to mutations in an unidentified gene(s) other than RET and VHL.

Adrenal Gland Neoplasms↗
PubMed2025

Mutational scanning of TnpB reveals latent activity for genome editing.

TnpB is a diverse family of RNA-guided endonucleases associated with prokaryotic transposons. Due to their small size and putative evolutionary relationship to CRISPR-Cas12, TnpB enzymes hold significant potential for genome editing. However, most TnpBs lack robust gene editing activity, and unbiased profiling of mutational effects on editing activity has not been explored. Here, we mapped comprehensive sequence-function landscapes of a TnpB ribonucleoprotein and discovered many activating mutations in both the protein and RNA. One- and two-position RNA mutants outperform existing variants, highlighting the utility of systematic RNA scaffold mutagenesis. Leveraging the protein's mutational landscape, we identified enhanced TnpB variants from a combinatorial library of activating mutations. These variants enhanced editing in human cells, N. benthamiana, pepper, and rice, with up to a fifty-fold increase compared to wild-type TnpB. These findings highlight previously unknown elements critical for regulating TnpB endonuclease activity and reveal surprising latent activity accessible through mutation.

Journal Article↗
PubMed1996

No mutations found by RET mutation scanning in sporadic and hereditary neuroblastoma.

Neuroblastoma occasionally occurs in diseases associated with abnormal neurocrest differentiation, e.g. Hirschsprung disease. Expression studies in developing mice suggest that the proto-oncogene RET plays a role in neurocrest differentiation. In humans expression of RET is limited to certain tumor types, including neuroblastoma, that derive from migrating neural crest cells. Mutations of RET are found associated with Hirschsprung disease. These data prompted us to investigate expression of RET and to search for gene mutations in neuroblastoma. Out of 16 neuroblastoma cell lines analyzed, 9 show clear expression of RET in a Northern blot analysis. In a single strandt conformation polymorphism (SSCP) analysis of all exons, no mutations were detected other than neutral polymorphisms. In a patient with neuroblastoma, from a family in which different neurocrestopathies, including neuroblastoma and Hirschsprung disease, had occurred, we also failed to detect RET mutations. Possibly, expression of RET in neuroblastoma merely reflects the differentiation status of the tumor cells. The absence of mutations suggests that RET does not play a crucial role in the tumorigenesis of neuroblastoma.

Drosophila Proteins↗