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Cold Spring Harbor Protocols Aug 2018Overlap extension polymerase chain reaction (PCR) mutagenesis can be used for the generation of a specific point mutation, insertion, or deletion within a particular DNA...
Overlap extension polymerase chain reaction (PCR) mutagenesis can be used for the generation of a specific point mutation, insertion, or deletion within a particular DNA sequence of interest. It requires relatively little preparation compared with other mutagenesis methods and does not require the use of restriction enzymes. Because of its versatility, the method has become widely used. Unlike methods of random mutagenesis, directed mutagenesis requires that the researcher already have a specific mutation in mind to implement. Traditional overlap extension PCR mutagenesis protocols remain limited in several critical ways, especially when it comes to generating insertions and deletions. For example, traditional protocols require that all sequence alterations be embedded within the primer itself, which makes it difficult to make insertions >30 nt. This protocol describes an overlap extension PCR mutagenesis method that is more versatile than its predecessors. Using this method, one can essentially make insertions and deletions of any size at any position within a given DNA sequence. To generate an insertion mutation, first prepare an insertion fragment and two flanking fragments by PCR. In the secondary PCR, the insertion fragment is recombined with two flanking fragments derived from the original template. This method can also be used to generate deletions, which is discussed in the latter part of the protocol.
Topics: DNA Primers; Mutagenesis, Insertional; Polymerase Chain Reaction; Sequence Deletion
PubMed: 30068588
DOI: 10.1101/pdb.prot097758 -
No Shinkei Geka. Neurological Surgery Sep 2023Meningioma is considered as a single type by the World Health Organization 2021 classification, encompassing 15 subtypes. These classifications are primarily based on...
Meningioma is considered as a single type by the World Health Organization 2021 classification, encompassing 15 subtypes. These classifications are primarily based on histological attributes. However, recent advancements in understanding driver gene mutations and molecular prognostic markers are of particular importance for comprehending the clinical behavior of these tumors. Meningiomas are stratified molecularly, predominantly based on the presence of NF2 mutation. Over 50% are associated with mutations in the NF2 gene and/or monosomy of chromosome 22, whereas the remaining are associated with gene mutations in either of AKT1, SMO, KLF4, TRAF7, or the other less common ones. Importantly, these driver gene mutations are mutually exclusive and significantly associated with tumor location, histological subtype, and grading. The majority of higher-grade meningiomas is associated with multiple chromosomal aberrations, including the loss of 1p, 6q, and 14q, along with NF2 mutations. Homozygous deletion of CDKN2A/CDKN2B genes or TERT promoter mutations serves as a predictor of poor prognosis. Notably, these molecular markers are now incorporated into the criteria for anaplastic meningioma. The amalgamation of molecular insights with clinical and histological parameters, alongside patient prognosis, holds significant utility in the management of patients with meningiomas. It is anticipated to pave the way for treatments founded on molecular-clinical associations.
Topics: Humans; Meningioma; Homozygote; Sequence Deletion; Mutation; Meningeal Neoplasms
PubMed: 37743334
DOI: 10.11477/mf.1436204824 -
Genetics in Medicine : Official Journal... Dec 2019Mediator is a multiprotein complex that allows the transfer of genetic information from DNA binding proteins to the RNA polymerase II during transcription initiation....
PURPOSE
Mediator is a multiprotein complex that allows the transfer of genetic information from DNA binding proteins to the RNA polymerase II during transcription initiation. MED12L is a subunit of the kinase module, which is one of the four subcomplexes of the mediator complex. Other subunits of the kinase module have been already implicated in intellectual disability, namely MED12, MED13L, MED13, and CDK19.
METHODS
We describe an international cohort of seven affected individuals harboring variants involving MED12L identified by array CGH, exome or genome sequencing.
RESULTS
All affected individuals presented with intellectual disability and/or developmental delay, including speech impairment. Other features included autism spectrum disorder, aggressive behavior, corpus callosum abnormality, and mild facial morphological features. Three individuals had a MED12L deletion or duplication. The other four individuals harbored single-nucleotide variants (one nonsense, one frameshift, and two splicing variants). Functional analysis confirmed a moderate and significant alteration of RNA synthesis in two individuals.
CONCLUSION
Overall data suggest that MED12L haploinsufficiency is responsible for intellectual disability and transcriptional defect. Our findings confirm that the integrity of this kinase module is a critical factor for neurological development.
Topics: Adolescent; Autism Spectrum Disorder; Child; Child, Preschool; Developmental Disabilities; Exome; Female; Frameshift Mutation; Humans; Intellectual Disability; Male; Mediator Complex; Mutation; Sequence Deletion; Transcription Factors; Young Adult
PubMed: 31155615
DOI: 10.1038/s41436-019-0557-3 -
Structure (London, England : 1993) Jun 2023In-frame deletion mutations can result in disease. The impact of these mutations on protein structure and subsequent functional changes remain understudied, partially...
In-frame deletion mutations can result in disease. The impact of these mutations on protein structure and subsequent functional changes remain understudied, partially due to the lack of comprehensive datasets including a structural readout. In addition, the recent breakthrough in structure prediction through deep learning demands an update of computational deletion mutation prediction. In this study, we deleted individually every residue of a small α-helical sterile alpha motif domain and investigated the structural and thermodynamic changes using 2D NMR spectroscopy and differential scanning fluorimetry. Then, we tested computational protocols to model and classify observed deletion mutants. We show a method using AlphaFold2 followed by RosettaRelax performs the best overall. In addition, a metric containing pLDDT values and Rosetta ΔΔG is most reliable in classifying tolerated deletion mutations. We further test this method on other datasets and show they hold for proteins known to harbor disease-causing deletion mutations.
Topics: Proteins; Mutation; Computer Simulation; Sequence Deletion; Magnetic Resonance Spectroscopy; Computational Biology
PubMed: 37119820
DOI: 10.1016/j.str.2023.04.005 -
Microbiology Spectrum Jun 2022The evolutional process of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) development remains inconclusive. This study compared the genome sequences of...
The evolutional process of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) development remains inconclusive. This study compared the genome sequences of severe acute respiratory syndrome coronavirus (SARS-CoV), bat coronavirus RaTG13, and SARS-CoV-2. In total, the genomes of SARS-CoV-2 and RaTG13 were 77.9% and 77.7% identical to the genome of SARS-CoV, respectively. A total of 3.6% (1,068 bases) of the SARS-CoV-2 genome was derived from insertion and/or deletion (indel) mutations, and 18.6% (5,548 bases) was from point mutations from the genome of SARS-CoV. At least 35 indel sites were confirmed in the genome of SARS-CoV-2, in which 17 were with ≥10 consecutive bases long. Ten of these relatively long indels were located in the spike (S) gene, five in nonstructural protein 3 (Nsp3) gene of open reading frame (ORF) 1a, and one in ORF8 and noncoding region. Seventeen (48.6%) of the 35 indels were based on insertion-and-deletion mutations with exchanged gene sequences of 7-325 consecutive bases. Almost the complete ORF8 gene was replaced by a single 325 consecutive base-long indel. The distribution of these indels was roughly in accordance with the distribution of the rate of point mutation rate around the indels. The genome sequence of SARS-CoV-2 was 96.0% identical to that of RaTG13. There was no long insertion-and-deletion mutation between the genomes of RaTG13 and SARS-CoV-2. The findings of the uneven distribution of multiple indels and the presence of multiple long insertion-and-deletion mutations with exchanged consecutive base sequences in the viral genome may provide insights into SARS-CoV-2 development. The developmental mechanism of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remains inconclusive. This study compared the base sequence one-by-one between severe acute respiratory syndrome coronavirus (SARS-CoV) or bat coronavirus RaTG13 and SARS-CoV-2. The genomes of SARS-CoV-2 and RaTG13 were 77.9% and 77.7% identical to the genome of SARS-CoV, respectively. Seventeen of the 35 sites with insertion and/or deletion mutations between SARS-CoV-2 and SARS-CoV were based on insertion-and-deletion mutations with the replacement of 7-325 consecutive bases. Most of these long insertion-and-deletion sites were concentrated in the nonstructural protein 3 (Nsp3) gene of open reading frame (ORF) 1a, S1 domain of the spike protein, and ORF8 genes. Such long insertion-and-deletion mutations were not observed between the genomes of RaTG13 and SARS-CoV-2. The presence of multiple long insertion-and-deletion mutations in the genome of SARS-CoV-2 and their uneven distributions may provide further insights into the development of the virus.
Topics: Animals; COVID-19; Chiroptera; Genome, Viral; Phylogeny; SARS-CoV-2; Sequence Deletion
PubMed: 35658573
DOI: 10.1128/spectrum.00716-22 -
Methods in Molecular Biology (Clifton,... 2023Mitochondrial DNA (mtDNA) deletions underpin mitochondrial dysfunction in human tissues in aging and disease. The multicopy nature of the mitochondrial genome means...
Mitochondrial DNA (mtDNA) deletions underpin mitochondrial dysfunction in human tissues in aging and disease. The multicopy nature of the mitochondrial genome means these mtDNA deletions can occur in varying mutation loads. At low levels, these deletions have no impact, but once the proportion of molecules harbouring a deletion exceeds a threshold level, then dysfunction occurs. The location of the breakpoints and the size of the deletion impact upon the mutation threshold required to cause deficiency of an oxidative phosphorylation complex, and this varies for each of the different complexes. Furthermore, mutation load and deletion species can vary between adjacent cells in a tissue, with a mosaic pattern of mitochondrial dysfunction observed. As such, it is often important for understanding human aging and disease to be able to characterise the mutation load, breakpoints and size of deletion(s) from a single human cell. Here, we detail protocols for laser micro-dissection and single cell lysis from tissues, and the subsequent analysis of deletion size, breakpoints and mutation load using long-range PCR, mtDNA sequencing and real-time PCR, respectively.
Topics: Humans; DNA, Mitochondrial; Aging; Mitochondria; Real-Time Polymerase Chain Reaction; Single-Cell Analysis; Sequence Deletion
PubMed: 36807808
DOI: 10.1007/978-1-0716-2922-2_29 -
Methods in Molecular Biology (Clifton,... 2023Mitochondrial DNA (mtDNA) mutations are found in several human pathologies and are associated with aging. Deletion mutations in mtDNA result in the loss of essential...
Mitochondrial DNA (mtDNA) mutations are found in several human pathologies and are associated with aging. Deletion mutations in mtDNA result in the loss of essential genes for mitochondrial function. Over 250 deletion mutations have been reported and the common deletion is the most frequent mtDNA deletion linked to disease. This deletion removes 4977 base pairs of mtDNA. It has previously been shown that exposure to UVA radiation can promote the formation of the common deletion. Furthermore, aberrations in mtDNA replication and repair are associated with formation of the common deletion. However, molecular mechanisms describing the formation of this deletion are poorly characterized. This chapter describes a method to irradiate human skin fibroblasts with physiological doses of UVA and the subsequent detection of the common deletion by quantitative PCR analysis.
Topics: Humans; DNA, Mitochondrial; Sequence Deletion; Mitochondria; Aging; Mutation
PubMed: 36807799
DOI: 10.1007/978-1-0716-2922-2_20 -
Human Molecular Genetics Aug 2018The co-occurrence of a copy number variant (CNV) and a functional variant on the other allele may be a relevant genetic mechanism in schizophrenia. We hypothesized that...
The co-occurrence of a copy number variant (CNV) and a functional variant on the other allele may be a relevant genetic mechanism in schizophrenia. We hypothesized that the cumulative burden of such double hits-in particular those composed of a deletion and a coding single-nucleotide variation (SNV)-is increased in patients with schizophrenia. We combined CNV data with coding variants data in 795 patients with schizophrenia and 474 controls. To limit false CNV-detection, only CNVs called by two algorithms were included. CNV-affected genes were subsequently examined for coding SNVs, which we termed "CNV-SNVs." Correcting for total queried sequence, we assessed the CNV-SNV-burden and the combined predicted deleterious effect. We estimated P-values by permutation of the phenotype. We detected 105 CNV-SNVs; 67 in duplicated and 38 in deleted genic sequence. Although the difference in CNV-SNVs rates was not significant, the combined deleteriousness inferred by CNV-SNVs in deleted sequence was almost 4-fold higher in cases compared with controls (nominal P = 0.009). This effect may be driven by a higher number of CNV-SNVs and/or by a higher degree of predicted deleteriousness of CNV-SNVs. No such effect was observed for duplications. We provide early evidence that deletions co-occurring with a functional variant may be relevant, albeit of modest impact, for the genetic etiology of schizophrenia. Large-scale consortium studies are required to validate our findings. Sequence-based analyses would provide the best resolution for detection of CNVs as well as coding variants genome-wide.
Topics: Case-Control Studies; DNA Copy Number Variations; Female; Humans; Male; Phenotype; Point Mutation; Polymorphism, Single Nucleotide; Schizophrenia; Sequence Deletion
PubMed: 29767709
DOI: 10.1093/hmg/ddy175 -
Analytical Biochemistry Mar 2021Deletion mutation has been proved as the important factor for occurrence and development of disease, especially those with cancer. With the popularity of precision...
Deletion mutation has been proved as the important factor for occurrence and development of disease, especially those with cancer. With the popularity of precision medicine, the individual cancer therapeutic strategy has highlighted the requirement to develop a straightforward and competent strategy for deletion mutation determination. Hence, the present study is dedicated to develop a one-step assay to identify deletion mutation with sequence specificity for clinical practice. Taking advantage of loop-mediated isothermal amplification, an ultrasensitive and rapid deletion mutation determination method is established, which allow as low as 30 copies or 0.1% target variants under strong interferential background can be accurately distinguished in 30 min dispensing with professional operation and complex data interpretation. As a demonstration, the epidermal growth factor receptor p.E746-A750del, a crucial factor for the susceptibility of tyrosine kinase inhibitor in non-small-cell lung cancer treatment, has been accurately identified by this method with both cell lines and real clinical samples. By tailor-made primer set, this method can be extended for other deletion mutants, making it a molecular diagnostic tool and could be readily adapted for cancer diagnosis, therapy and prognosis in point of care diagnostic test scenario.
Topics: Biomarkers, Tumor; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; DNA Mutational Analysis; DNA Primers; ErbB Receptors; Humans; Lung Neoplasms; Molecular Diagnostic Techniques; Nucleic Acid Amplification Techniques; Sensitivity and Specificity; Sequence Deletion
PubMed: 33352189
DOI: 10.1016/j.ab.2020.114087 -
Reproductive Sciences (Thousand Oaks,... Feb 2022Acephalic spermatozoa syndrome (ASS) is a severe form of teratozoospermia, previous studies have shown that SUN5 mutations are the major cause of acephalic spermatozoa...
Acephalic spermatozoa syndrome (ASS) is a severe form of teratozoospermia, previous studies have shown that SUN5 mutations are the major cause of acephalic spermatozoa syndrome. This study is to identify the pathogenic mutations in SUN5 leading to ASS. PCR and Sanger sequence were performed to define the breakpoints and mutations in SUN5. Whole genome sequencing (WGS) was performed to detect heterozygous deletion. Western blotting and immunofluorescence analysis detected the expression level and localization of SUN5. Furthermore, the pathogenicity of the mutant SUN5 was predicted in silico and was verified by the experiments in vitro. We identified one novel homozygous missense mutation (c.775G>A; p.G259S) and one compound heterozygous including one reported missense mutation (c.1043A>T; p.N348I) and a large deletion that contains partial EFCAB8 ( NM_001143967 .1) and BPIFB2 ( NM_025227 ) and complete SUN5 ( NM_080675 ), and one recurrent homozygous splice-site mutation (c.340G>A; p.G114R) in SUN5 in three patients with ASS. Our results showed that SUN5 could not be detected in the patients' spermatozoa and the exogenous expression level of the mutant protein was decreased in transfected HEK-293T cells. This study expands the mutational spectrum of SUN5. We recommended a clinical diagnostic strategy for SUN5 genomic deletion to screen heterozygous deletions and indicated that the diagnostic value of screening for SUN5 mutations and deletions in infertile men with ASS.
Topics: Adult; Blotting, Western; Fluorescent Antibody Technique; HEK293 Cells; Humans; Infertility, Male; Male; Membrane Proteins; Mutation, Missense; Pedigree; Sequence Deletion; Spermatozoa; Syndrome; Teratozoospermia; Whole Genome Sequencing
PubMed: 34159570
DOI: 10.1007/s43032-021-00665-5