-
BMC Biotechnology Dec 2008Mutagenesis plays an essential role in molecular biology and biochemistry. It has also been used in enzymology and protein science to generate proteins which are more...
BACKGROUND
Mutagenesis plays an essential role in molecular biology and biochemistry. It has also been used in enzymology and protein science to generate proteins which are more tractable for biophysical techniques. The ability to quickly and specifically mutate a residue(s) in protein is important for mechanistic and functional studies. Although many site-directed mutagenesis methods have been developed, a simple, quick and multi-applicable method is still desirable.
RESULTS
We have developed a site-directed plasmid mutagenesis protocol that preserved the simple one step procedure of the QuikChange site-directed mutagenesis but enhanced its efficiency and extended its capability for multi-site mutagenesis. This modified protocol used a new primer design that promoted primer-template annealing by eliminating primer dimerization and also permitted the newly synthesized DNA to be used as the template in subsequent amplification cycles. These two factors we believe are the main reasons for the enhanced amplification efficiency and for its applications in multi-site mutagenesis.
CONCLUSION
Our modified protocol significantly increased the efficiency of single mutation and also allowed facile large single insertions, deletions/truncations and multiple mutations in a single experiment, an option incompatible with the standard QuikChange. Furthermore the new protocol required significantly less parental DNA which facilitated the DpnI digestion after the PCR amplification and enhanced the overall efficiency and reliability. Using our protocol, we generated single site, multiple single-site mutations and a combined insertion/deletion mutations. The results demonstrated that this new protocol imposed no additional reagent costs (beyond basic QuikChange) but increased the overall success rates.
Topics: DNA Primers; Mutagenesis, Insertional; Mutagenesis, Site-Directed; Plasmids; Polymerase Chain Reaction; Sequence Deletion
PubMed: 19055817
DOI: 10.1186/1472-6750-8-91 -
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 -
GeroScience Jun 2021Mitochondrial DNA (mtDNA) quality and quantity relate to two hallmarks of aging-genomic instability and mitochondrial dysfunction. Physical performance relies on...
Mitochondrial DNA (mtDNA) quality and quantity relate to two hallmarks of aging-genomic instability and mitochondrial dysfunction. Physical performance relies on mitochondrial integrity and declines with age, yet the interactions between mtDNA quantity, quality, and physical performance are unclear. Using a validated digital PCR assay specific for mtDNA deletions, we tested the hypothesis that skeletal muscle mtDNA deletion mutation frequency (i.e., a measure of mtDNA quality) or mtDNA copy number predicts physical performance in older adults. Total DNA was isolated from vastus lateralis muscle biopsies and used to quantitate mtDNA copy number and mtDNA deletion frequency by digital PCR. The biopsies were obtained from a cross-sectional cohort of 53 adults aged 50 to 86 years. Before the biopsy procedure, physical performance measurements were collected, including VO, modified physical performance test score, 6-min walk distance, gait speed, grip strength, and total lean and leg mass. Linear regression models were used to evaluate the relationships between age, sex, and the outcomes. We found that mtDNA deletion mutation frequency increased exponentially with advancing age. On average from ages 50 to 86, deletion frequency increased from 0.008 to 0.15%, an 18-fold increase. Females may have lower deletion frequencies than males at older ages. We also measured declines in VO and mtDNA copy number with age in both sexes. The mtDNA deletion frequency measured from single skeletal muscle biopsies predicted 13.3% of the variation in VO. Copy number explained 22.6% of the variation in mtDNA deletion frequency and 10.4% of the lean mass variation. We found predictive relationships between age, mtDNA deletion mutation frequency, mtDNA copy number, and physical performance. These data are consistent with a role for mitochondrial function and genome integrity in maintaining physical performance with age. Analyses of mtDNA quality and quantity in larger cohorts and longitudinal studies could extend our understanding of the importance of mitochondrial DNA in human aging and longevity.
Topics: Aged; Aged, 80 and over; Cross-Sectional Studies; DNA Copy Number Variations; DNA, Mitochondrial; Female; Humans; Male; Middle Aged; Mitochondria; Muscle, Skeletal; Physical Functional Performance; Sequence Deletion
PubMed: 33740224
DOI: 10.1007/s11357-021-00351-z -
Aging Cell Jun 2023Mitochondrial DNA (mtDNA) deletion mutations cause many human diseases and are linked to age-induced mitochondrial dysfunction. Mapping the mutation spectrum and...
Mitochondrial DNA (mtDNA) deletion mutations cause many human diseases and are linked to age-induced mitochondrial dysfunction. Mapping the mutation spectrum and quantifying mtDNA deletion mutation frequency is challenging with next-generation sequencing methods. We hypothesized that long-read sequencing of human mtDNA across the lifespan would detect a broader spectrum of mtDNA rearrangements and provide a more accurate measurement of their frequency. We employed nanopore Cas9-targeted sequencing (nCATS) to map and quantitate mtDNA deletion mutations and develop analyses that are fit-for-purpose. We analyzed total DNA from vastus lateralis muscle in 15 males ranging from 20 to 81 years of age and substantia nigra from three 20-year-old and three 79-year-old men. We found that mtDNA deletion mutations detected by nCATS increased exponentially with age and mapped to a wider region of the mitochondrial genome than previously reported. Using simulated data, we observed that large deletions are often reported as chimeric alignments. To address this, we developed two algorithms for deletion identification which yield consistent deletion mapping and identify both previously reported and novel mtDNA deletion breakpoints. The identified mtDNA deletion frequency measured by nCATS correlates strongly with chronological age and predicts the deletion frequency as measured by digital PCR approaches. In substantia nigra, we observed a similar frequency of age-related mtDNA deletions to those observed in muscle samples, but noted a distinct spectrum of deletion breakpoints. NCATS-mtDNA sequencing allows the identification of mtDNA deletions on a single-molecule level, characterizing the strong relationship between mtDNA deletion frequency and chronological aging.
Topics: Male; Humans; Nanopore Sequencing; Sequence Deletion; Aging; Longevity; DNA, Mitochondrial
PubMed: 37132288
DOI: 10.1111/acel.13842 -
Archives of Microbiology Mar 2023The insertion/deletion (indel) mutation profiles of SARS-CoV-2 variants, including Omicron, remain unclear. We compared whole-genome sequences from various lineages and...
The insertion/deletion (indel) mutation profiles of SARS-CoV-2 variants, including Omicron, remain unclear. We compared whole-genome sequences from various lineages and used preserved indels to infer the ancestral relationships between different lineages. Thirteen indel patterns from twelve sites were seen in ≥ 2 sequences; six of these sites were located in the N-terminal domain of the viral spike gene. Preserved indels in the coding regions were also identified in the non-structural protein 3 (Nsp3), Nsp6, and nucleocapsid genes. Seven of the thirteen indel patterns were specific to the Omicron variants, four of which were observed in BA.1, making it the most mutated variant. Other preserved indels observed in the Omicron variants were also seen in Alpha and/or Gamma, but not Delta, suggesting that Omicron is phylogenetically more proximal to Alpha. We demonstrated distinct profiles of preserved indels among SARS-CoV-2 variants and sublineages, suggesting the importance of indels in viral evolution.
Topics: Humans; SARS-CoV-2; COVID-19; Gamma Rays; Sequence Deletion
PubMed: 37000302
DOI: 10.1007/s00203-023-03493-0 -
Ecotoxicology and Environmental Safety Jun 2023Due to the rapid production growth and a wide range of applications, safety concerns are being raised about the genotoxic properties of silver nanoparticles (AgNPs). In...
Due to the rapid production growth and a wide range of applications, safety concerns are being raised about the genotoxic properties of silver nanoparticles (AgNPs). In this research, we found AgNPs induced a size-dependent genotoxicity via lysosomal-autophagy dysfunction in human-hamster hybrid (A) cells. Compared with 25 nm and 75 nm particles, 5 nm AgNPs could accentuate the genotoxic responses, including DNA double-strand breaks (DSBs) and multi-locus deletion mutation, which could be significantly enhanced by autophagy inhibitors 3-methyl adenine (3-MA), Bafilomycin A1 (BFA), and cathepsin inhibitors, respectively. The autophagy dysfunction was closely related to the accumulation of 5 nm AgNPs in the lysosomes and the interruption of lysosome-autophagosome fusion. With lysosomal protective agent 3-O-Methylsphingomyelin (3-O-M) and endocytosis inhibitor wortmannin, the reactivation of lysosomal function and the recovery of autophagy significantly attenuated AgNP-induced genotoxicity. Our data provide clear evidence to illustrate the role of subcellular targets in the genotoxicity of AgNPs in mammalian cells, which laid the basis for better understanding the health risk of AgNPs and their related products.
Topics: Animals; Humans; Silver; Metal Nanoparticles; Autophagy; Lysosomes; Sequence Deletion; Mammals
PubMed: 37105094
DOI: 10.1016/j.ecoenv.2023.114947 -
Journal of Clinical Laboratory Analysis Sep 2019Hb H disease is a serious type of α-thalassemia which cause moderate anemia while misdiagnosis by routine genetic analysis in a rare or novel Hb H disease.
BACKGROUND
Hb H disease is a serious type of α-thalassemia which cause moderate anemia while misdiagnosis by routine genetic analysis in a rare or novel Hb H disease.
METHODS
The study was done on three patients and one fetus in a suspected Hb H disease family. Hb analysis was carried out using capillary electrophoresis (CE), and hematological analysis was conducted with an automated cell counter. Common α- and β-thalassemia mutations were detected by routine genetic analysis (gap-PCR and RDB-PCR). Novel mutation diagnostic methods were based on DNA sequencing.
RESULTS
Capillary electrophoresis revealed clinical feature of classic Hb H disease in the proband, and hematology analysis showed moderate anemia (Hb 87 g/L). But routine genetic analysis was found that it was only a heterozygote for the -- deletion. DNA sequencing of α-globin genes (α1 and α2) identified the breakpoints between nts 34162 and 34171 at α2 gene, named CD 90-93 (-AGCTTCGG) mutation. The genotype of proband and fetus was the same -- /-α . His father was homozygous for the novel mutation (-α /-α ), and his mother was heterozygote for the -- deletion.
CONCLUSIONS
Our study for the first time described the novel mutation CD 90-93 (-AGCTTCGG). CE is a way to avoid misdiagnosis of rare or novel Hb H disease.
Topics: Base Sequence; Child, Preschool; Electrophoresis, Capillary; Hemoglobins, Abnormal; Humans; Male; Mutation; Sequence Analysis, DNA; Sequence Deletion; alpha-Thalassemia
PubMed: 31199523
DOI: 10.1002/jcla.22949 -
Molecular Genetics & Genomic Medicine May 2023Pseudohypoparathyroidism (PHP) is a series of diseases related to pathological changes and neurocognitive and endocrine abnormalities, mainly due to the GNAS mutation on...
BACKGROUND
Pseudohypoparathyroidism (PHP) is a series of diseases related to pathological changes and neurocognitive and endocrine abnormalities, mainly due to the GNAS mutation on chromosome 20q13.2, which weakens receptor-mediated hormone signal transduction. Considering its complex genetic and epigenetic characteristics, GNAS may produce complex clinical phenotypes in families or sporadic cases. This study presented a case of familial PHP caused by a deletion mutation in the 20q13.2 region.
METHODS AND RESULTS
The proband and her second daughter had PHP, and the proband's mother had pseudo-PHP. Whole-genome sequencing revealed that the proband had an 849.81 kb deletion spanning GNAS near the maternal 20q13.2 chromosome. Multiplex ligation-dependent probe amplification methylation analysis indicated that the proband as well as her mother and second daughter had seemingly abnormal GNAS methylation. This is different from the phenotype (feeding difficulty, slow growth, and special facial features) of previously reported cases with the deletion of fragments near the 20q13.2 chromosome.
CONCLUSIONS
This report demonstrated the variability of 20q13.2 deletion phenotypes and the clinical importance of using multiple molecular genetic detection methods.
Topics: Female; Humans; GTP-Binding Protein alpha Subunits, Gs; DNA Methylation; Chromogranins; Pseudohypoparathyroidism; Sequence Deletion
PubMed: 36669868
DOI: 10.1002/mgg3.2144 -
Orphanet Journal of Rare Diseases Jul 2018Dystrophinopathies are a set of severe and incurable X-linked neuromuscular disorders caused by mutations in the dystrophin gene (DMD). These mutations form a complex...
BACKGROUND
Dystrophinopathies are a set of severe and incurable X-linked neuromuscular disorders caused by mutations in the dystrophin gene (DMD). These mutations form a complex spectrum. A national registration network is essential not only to provide more information about the prevalence and natural history of the disease, but also to collect genetic data for analyzing the mutational spectrum. This information is extremely beneficial for basic scientific research, genetic diagnosis, trial planning, clinical care, and gene therapy.
METHODS
We collected data from 1400 patients (1042 patients with confirmed unrelated Duchenne muscular dystrophy [DMD] or Becker muscular dystrophy [BMD]) registered in the Chinese Genetic Disease Registry from March 2012 to August 2017 and analyzed the genetic mutational characteristics of these patients.
RESULTS
Large deletions were the most frequent type of mutation (72.2%), followed by nonsense mutations (11.9%), exon duplications (8.8%), small deletions (3.0%), splice-site mutations (2.1%), small insertions (1.3%), missense mutations (0.6%), and a combination mutation of a deletion and a duplication (0.1%). Exon 45-50 deletion was the most frequent deletion type, while exon 2 duplication was the most common duplication type. Two deletion hotspots were calculated-one located toward the central part (exon 45-52) of the gene and the other toward the 5'end (exon 8-26). We found no significant difference between hereditary and de novo mutations on deletion hotspots. Nonsense mutations accounted for 62.9% of all small mutations (197 patients).
CONCLUSION
We built a comprehensive national dystrophinopathy mutation database in China, which is essential for basic and clinical research in this field. The mutational spectrum and characteristics of this DMD/BMD group were largely consistent with those in previous international DMD/BMD studies, with some differences. Based on our results, about 12% of DMD/BMD patients with nonsense mutations may benefit from stop codon read-through therapy. Additionally, the top three targets for exon-skipping therapy are exon 51 (141, 13.5%), exon 53 (115, 11.0%), and exon 45 (84, 8.0%).
Topics: China; Codon, Nonsense; Dystrophin; Exons; Humans; Muscular Dystrophy, Duchenne; Mutation; Mutation, Missense; Sequence Deletion
PubMed: 29973226
DOI: 10.1186/s13023-018-0853-z -
Molecular Genetics & Genomic Medicine Mar 2021Netherton syndrome (NS) is an autosomal recessive disorder due to mutations in the SPINK5 gene. Here, we report the first case of NS caused by a large genomic deletion.
Netherton syndrome caused by compound heterozygous mutation, c.80A>G mutation in SPINK5 and large-sized genomic deletion mutation, and successful treatment of intravenous immunoglobulin.
BACKGROUND
Netherton syndrome (NS) is an autosomal recessive disorder due to mutations in the SPINK5 gene. Here, we report the first case of NS caused by a large genomic deletion.
METHODS
We present the clinical data of a 3-year-old Chinese boy who was initially misdiagnosed with severe atopic dermatitis. Subsequently, the patient presented with typical ichthyosis linearis circumflexa and had representative hair shaft of trichorrhexis invaginate, which alerted the physician of the high possibility of NS. A genomic DNA sample was extracted from peripheral blood and whole-exome sequencing (WES) was performed. Sanger sequencing and quantitative real-time polymerase chain reaction (qRT-PCR) were performed to verify the mutation and genomic deletion, respectively, in the pedigree.
RESULTS
WES revealed compound heterozygous mutations in SPINK5, including a c.80A>G mutation and a ~275 Kb-sized genomic deletion (chr5:147443576-147719312). The c.80A>G mutation was verified by Sanger sequencing in the pedigree. The father had the same heterozygous mutation; however, the mutation was absent in the proband's mother. The qRT-PCR results identified a large deletion (chr5:147444834-147445034) in SPINK5 in the proband and his mother. The eruptions improved remarkably after intravenous immunoglobulin (IVIG) therapy.
CONCLUSIONS
This is the first observation of NS caused by a large deletion. Our findings have important implications for mutation screening and genetic counseling in NS. Our report also verifies and supports the safety and efficacy of IVIG therapy in patients with NS.
Topics: Adult; Child, Preschool; Female; Heterozygote; Humans; Immunoglobulins, Intravenous; Male; Netherton Syndrome; Point Mutation; Sequence Deletion; Serine Peptidase Inhibitor Kazal-Type 5
PubMed: 33452875
DOI: 10.1002/mgg3.1600