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Methods in Molecular Biology (Clifton,... 2017Inverse PCR is a powerful tool for the rapid introduction of desired mutations at desired positions in a circular double-stranded DNA sequence. Here, custom-designed...
Inverse PCR is a powerful tool for the rapid introduction of desired mutations at desired positions in a circular double-stranded DNA sequence. Here, custom-designed mutant primers oriented in the inverse direction are used to amplify the entire circular template with incorporation of the required mutation(s). By careful primer design it can be used to perform such diverse modifications as the introduction of point mutations and multiple mutations, the insertion of new sequences, and even sequence deletions. Three primer formats are commonly used; nonoverlapping, partially overlapping and fully overlapping primers, and here we describe the use of nonoverlapping primers for introduction of a point mutation. Use of such a primer setup in the PCR reaction, with one of the primers containing the desired mismatch mutation, results in the amplification of a linear, double-stranded, mutated product. Methylated template DNA is removed from the nonmethylated PCR product by DpnI digestion and the PCR product is then phosphorylated by polynucleotide kinase treatment before being recircularized by ligation, and transformed to E. coli. This relatively simple site-directed mutagenesis procedure is of major importance in biology and biotechnology today where it is commonly employed for the study and engineering of DNA, RNA, and proteins.
Topics: DNA; DNA Primers; Mutagenesis, Site-Directed; Point Mutation; Polymerase Chain Reaction
PubMed: 28540701
DOI: 10.1007/978-1-4939-7060-5_5 -
Journal of Cell Science Apr 2022Coilin is a conserved protein essential for integrity of nuclear membrane-less inclusions called Cajal bodies. Here, we report an amino acid substitution (p.K496E) found...
Coilin is a conserved protein essential for integrity of nuclear membrane-less inclusions called Cajal bodies. Here, we report an amino acid substitution (p.K496E) found in a widely-used human EGFP-coilin construct that has a dominant-negative effect on Cajal body formation. We show that this coilin-K496E variant fails to rescue Cajal bodies in cells lacking endogenous coilin, whereas the wild-type construct restores Cajal bodies in mouse and human coilin-knockout cells. In cells containing endogenous coilin, both the wild-type and K496E variant proteins accumulate in Cajal bodies. However, high-level overexpression of coilin-K496E causes Cajal body disintegration. Thus, a mutation in the C-terminal region of human coilin can disrupt Cajal body assembly. Caution should be used when interpreting data from coilin plasmids that are derived from this variant (currently deposited at Addgene).
Topics: Animals; Coiled Bodies; HeLa Cells; Humans; Mice; Mutation; Nuclear Proteins; Point Mutation
PubMed: 35356988
DOI: 10.1242/jcs.259587 -
Nature Biomedical Engineering Jan 2020
Topics: Adenine; Animals; Mice; Point Mutation; Tyrosinemias
PubMed: 31937937
DOI: 10.1038/s41551-019-0489-x -
Gastrointestinal Endoscopy Mar 2021
Topics: Adenocarcinoma; Biomarkers; High-Throughput Nucleotide Sequencing; Humans; Pancreatic Neoplasms; Point Mutation
PubMed: 33583519
DOI: 10.1016/j.gie.2020.07.024 -
Disease Models & Mechanisms Oct 2018The zebrafish is an increasingly popular model organism for human genetic disease research. CRISPR/Cas9-based approaches are currently used for multiple gene-editing...
The zebrafish is an increasingly popular model organism for human genetic disease research. CRISPR/Cas9-based approaches are currently used for multiple gene-editing purposes in zebrafish, but few studies have developed reliable ways to introduce precise mutations. Point mutation knock-in using CRISPR/Cas9 and single-stranded oligodeoxynucleotides (ssODNs) is currently the most promising technology for this purpose. Despite some progress in applying this technique to zebrafish, there is still a great need for improvements in terms of its efficiency, optimal design of sgRNA and ssODNs and broader applicability. The papers discussed in this Editorial provide excellent case studies on identifying problems inherent in the mutation knock-in technique, quantifying these issues and proposing strategies to overcome them. These reports also illustrate how the procedures for introducing specific mutations can be straightforward, such that ssODNs with only the target mutation are sufficient for generating the intended knock-in animals. Two of the studies also develop interesting point mutant knock-in models for cardiac diseases, validating the translational relevance of generating knock-in mutations and opening the door to many possibilities for their further study.
Topics: Animals; CRISPR-Cas Systems; Disease Models, Animal; Gene Knock-In Techniques; Point Mutation; Zebrafish
PubMed: 30366936
DOI: 10.1242/dmm.037515 -
Activity and Point Mutation G699V in PcoORP1 Confer Resistance to Oxathiapiprolin in Field Isolates.Journal of Agricultural and Food... Nov 2022The oxysterol-binding protein inhibitor oxathiapiprolin is a new fungicide for controlling oomycetes diseases. Besides, laboratory mutagenesis oxathiapiprolin-resistance...
The oxysterol-binding protein inhibitor oxathiapiprolin is a new fungicide for controlling oomycetes diseases. Besides, laboratory mutagenesis oxathiapiprolin-resistance among phytopathogenic oomycetes in the field remains unknown. Here, the sensitivity of 97 isolates to oxathiapiprolin was examined that were collected between 2011 and 2016. We obtained a baseline sensitivity with a mean EC value of 5.2639 × 10 μg mL. We showed that 6/32 isolates collected in Fujian Province from 2019 to 2020 were resistant to oxathiapiprolin without a significant fitness penalty on sporulation, vegetative growth, and virulence of the field isolates. The oxathiapiprolin resistance field isolates contained the point mutation glycine to valine at 699 in . The point mutation G699V was verified to confer resistance of to oxathiapiprolin using the CRISPR/Cas9 system. The mutation G699V decreased the binding affinity between oxathiapiprolin and . These results will improve our understanding of the mechanism of resistance to oxathiapiprolin under field conditions.
Topics: Phytophthora; Point Mutation; Plant Diseases; Hydrocarbons, Fluorinated; Fungicides, Industrial
PubMed: 36315898
DOI: 10.1021/acs.jafc.2c06707 -
Current Opinion in Microbiology Oct 2001Low levels of recombination in bacterial species have often been inferred from the presence of linkage disequilibrium between the alleles at different loci in the... (Review)
Review
Low levels of recombination in bacterial species have often been inferred from the presence of linkage disequilibrium between the alleles at different loci in the population. However, significant linkage disequilibrium is inevitable in organisms that divide by binary fission, and recombinational replacements must be very frequent, compared to point mutation, to dissipate disequilibrium. Recent studies using data from multilocus sequence typing indicate that, in many species, recombinational replacements contribute more greatly to clonal diversification than do point mutations and, in some species, recombination has been sufficient to eliminate any phylogenetic signal from gene trees. Recent efforts to improve understanding of the extent and impact of homologous recombination in the diversification of bacterial clones are discussed.
Topics: Bacteria; Genetic Variation; Humans; Point Mutation; Recombination, Genetic
PubMed: 11587939
DOI: 10.1016/s1369-5274(00)00257-5 -
Cellular and Molecular Neurobiology Oct 2023The BAF (BRG1/BRM-associated factor) chromatin remodelling complex is essential for the regulation of DNA accessibility and gene expression during neuronal...
The BAF (BRG1/BRM-associated factor) chromatin remodelling complex is essential for the regulation of DNA accessibility and gene expression during neuronal differentiation. Mutations of its core subunit SMARCB1 result in a broad spectrum of pathologies, including aggressive rhabdoid tumours or neurodevelopmental disorders. Other mouse models have addressed the influence of a homo- or heterozygous loss of Smarcb1, yet the impact of specific non-truncating mutations remains poorly understood. Here, we have established a new mouse model for the carboxy-terminal Smarcb1 c.1148del point mutation, which leads to the synthesis of elongated SMARCB1 proteins. We have investigated its impact on brain development in mice using magnetic resonance imaging, histology, and single-cell RNA sequencing. During adolescence, Smarcb1 mice demonstrated rather slow weight gain and frequently developed hydrocephalus including enlarged lateral ventricles. In embryonic and neonatal stages, mutant brains did not differ anatomically and histologically from wild-type controls. Single-cell RNA sequencing of brains from newborn mutant mice revealed that a complete brain including all cell types of a physiologic mouse brain is formed despite the SMARCB1 mutation. However, neuronal signalling appeared disturbed in newborn mice, since genes of the AP-1 transcription factor family and neurite outgrowth-related transcripts were downregulated. These findings support the important role of SMARCB1 in neurodevelopment and extend the knowledge of different Smarcb1 mutations and their associated phenotypes.
Topics: Animals; Mice; Hydrocephalus; Mutation; Point Mutation; Signal Transduction; Transcription Factor AP-1
PubMed: 37219662
DOI: 10.1007/s10571-023-01361-5 -
Homology directed correction, a new pathway model for point mutation repair catalyzed by CRISPR-Cas.Scientific Reports May 2022Gene correction is often referred to as the gold standard for precise gene editing and while CRISPR-Cas systems continue to expand the toolbox for clinically relevant...
Gene correction is often referred to as the gold standard for precise gene editing and while CRISPR-Cas systems continue to expand the toolbox for clinically relevant genetic repair, mechanistic hurdles still hinder widespread implementation. One of the most prominent challenges to precise CRISPR-directed point mutation repair centers on the prevalence of on-site mutagenesis, wherein insertions and deletions appear at the targeted site following correction. Here, we introduce a pathway model for Homology Directed Correction, specifically point mutation repair, which enables a foundational analysis of genetic tools and factors influencing precise gene editing. To do this, we modified an in vitro gene editing system which utilizes a cell-free extract, CRISPR-Cas RNP and donor DNA template to catalyze point mutation repair. We successfully direct correction of four unique point mutations which include two unique nucleotide mutations at two separate targeted sites and visualize the repair profiles resulting from these reactions. This extension of the cell-free gene editing system to model point mutation repair may provide insight for understanding the factors influencing precise point mutation correction.
Topics: CRISPR-Cas Systems; Catalysis; Gene Editing; Mutagenesis; Mutation; Point Mutation
PubMed: 35581233
DOI: 10.1038/s41598-022-11808-2 -
BMC Medical Genomics Jan 2021In addition to ovarian and breast cancers, loss-of-function mutations in BRCA1 and BRCA2 genes are also linked to an increased risk of pancreatic cancer, with ~ 4 to...
BACKGROUND
In addition to ovarian and breast cancers, loss-of-function mutations in BRCA1 and BRCA2 genes are also linked to an increased risk of pancreatic cancer, with ~ 4 to 7% of pancreatic cancer patients harboring germline BRCA mutations. Most BRCA alterations in pancreatic cancer are frame-shifting indels, stop-gain, and splice-site mutations, but single nucleotide substitutions are rare. Recent studies demonstrated a significant progression-free survival (PFS) benefit from maintenance olaparib, a poly (ADP-ribose) polymerase (PARP) inhibitor administered to patients with germline BRCA mutations and metastatic pancreatic cancer.
CASE PRESENTATION
Here, we report a metastatic pancreatic cancer case who harbored a novel somatic BRCA2 c.6944T > C (p. I2315T) point mutation. After 6 weeks first-line chemotherapy, the patient was refractory to treatment and had a progressive disease. Due to the novel nonsynonymous BRCA2 point mutation, we decided to change the strategy by administering olaparib. The patient benefited from olaparib therapy and achieved a PFS of ~ 6.5 months.
CONCLUSIONS
We describe a patient carrying a novel somatic BRCA2 p. I2315T point mutation, which is first reported in metastatic pancreatic cancer. This case report indicates that a gene mutation-based strategy should be considered in the clinic to provide more effective treatment.
Topics: Female; Genes, BRCA2; Humans; Pancreatic Neoplasms; Phthalazines; Piperazines; Point Mutation
PubMed: 33407459
DOI: 10.1186/s12920-020-00850-6