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Journal of Mass Spectrometry : JMS Feb 1998Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI/TOF-MS) of a restriction endonuclease digest determines the molecular mass of...
Separate analysis of complementary strands of restriction enzyme-digested DNA. An application of restriction fragment mass mapping by matrix-assisted laser desorption/ionization mass spectrometry.
Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI/TOF-MS) of a restriction endonuclease digest determines the molecular mass of PCR-amplified DNA more easily than measurement of undigested DNA. With this method, a 664 bp region from the FAS gene could be analyzed and a two-nucleotide deletion in the L1CAM gene was detected in a restriction fragment of 105 nucleotides. Furthermore, the analysis of smaller fragments allowed separate detection of single-stranded oligonucleotides comprising individual digested fragments. This mixture analysis of restriction enzyme digests improves the resolution, sensitivity and accuracy of MALDI/TOF-MS of DNA and is thus expected to facilitate its application to genetic diagnosis.
Topics: DNA; DNA Restriction Enzymes; Hydrolysis; Mutation; Polymerase Chain Reaction; Restriction Mapping; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
PubMed: 9487689
DOI: 10.1002/(SICI)1096-9888(199802)33:2<187::AID-JMS625>3.0.CO;2-A -
Methods in Molecular Biology (Clifton,... 1997
Topics: Biotin; Chromatin; Chromosome Mapping; DNA; DNA Probes; Digoxigenin; Humans; In Situ Hybridization, Fluorescence; Neoplasms; Polymerase Chain Reaction; Restriction Mapping
PubMed: 9055250
DOI: 10.1385/0-89603-482-8:53 -
Scientific Reports Dec 2017Next-generation sequencing (NGS) has caused a revolution, yet left a gap: long-range genetic information from native, non-amplified DNA fragments is unavailable. It...
Next-generation sequencing (NGS) has caused a revolution, yet left a gap: long-range genetic information from native, non-amplified DNA fragments is unavailable. It might be obtained by optical mapping of megabase-sized DNA molecules. Frequently only a specific genomic region is of interest, so here we introduce a method for selection and enrichment of megabase-sized DNA molecules intended for single-molecule optical mapping: DNA from a human cell line is digested by the NotI rare-cutting enzyme and size-selected by pulsed-field gel electrophoresis. For demonstration, more than 600 sub-megabase- to megabase-sized DNA molecules were recovered from the gel and analysed by denaturation-renaturation optical mapping. Size-selected molecules from the same gel were sequenced by NGS. The optically mapped molecules and the NGS reads showed enrichment from regions defined by NotI restriction sites. We demonstrate that the unannotated genome can be characterized in a locus-specific manner via molecules partially overlapping with the annotated genome. The method is a promising tool for investigation of structural variants in enriched human genomic regions for both research and diagnostic purposes. Our enrichment method could potentially work with other genomes or target specified regions by applying other genomic editing tools, such as the CRISPR/Cas9 system.
Topics: Chromosome Mapping; DNA; Electrophoresis, Gel, Pulsed-Field; Female; Genome, Human; Genomics; High-Throughput Nucleotide Sequencing; Humans; Restriction Mapping; Sequence Analysis, DNA
PubMed: 29263336
DOI: 10.1038/s41598-017-18091-6 -
Bioinformatics (Oxford, England) Apr 2016The Optical Mapping System discovers structural variants and potentiates sequence assembly of genomes via scaffolding and comparisons that globally validate or correct...
MOTIVATION
The Optical Mapping System discovers structural variants and potentiates sequence assembly of genomes via scaffolding and comparisons that globally validate or correct sequence assemblies. Despite its utility, there are few publicly available tools for aligning optical mapping datasets.
RESULTS
Here we present software, named 'Maligner', for the alignment of both single molecule restriction maps (Rmaps) and in silico restriction maps of sequence contigs to a reference. Maligner provides two modes of alignment: an efficient, sensitive dynamic programming implementation that scales to large eukaryotic genomes, and a faster indexed based implementation for finding alignments with unmatched sites in the reference but not the query. We compare our software to other publicly available tools on Rmap datasets and show that Maligner finds more correct alignments in comparable runtime. Lastly, we introduce the M-Score statistic for normalizing alignment scores across restriction maps and demonstrate its utility for selecting high quality alignments.
AVAILABILITY AND IMPLEMENTATION
The Maligner software is written in C ++ and is available at https://github.com/LeeMendelowitz/maligner under the GNU General Public License.
CONTACT
Topics: Algorithms; Computer Simulation; Genome; Restriction Mapping; Sequence Alignment; Sequence Analysis, DNA; Software
PubMed: 26637292
DOI: 10.1093/bioinformatics/btv711 -
Molecular Genetics & Genomic Medicine Mar 2019Facioscapulohumeral muscular dystrophy 1 (FSHD1) is a relatively common autosomal dominant adult muscular dystrophy with variable disease penetrance. The disease is...
INTRODUCTION
Facioscapulohumeral muscular dystrophy 1 (FSHD1) is a relatively common autosomal dominant adult muscular dystrophy with variable disease penetrance. The disease is caused by shortening of a D4Z4 repeat array located near the telomere of chromosome 4 at 4q35. This causes activation of a dormant gene DUX4, permitting aberrant DUX4 expression which is toxic to muscles. Molecular diagnosis of FSHD1 by Southern blot hybridization or FISH combing is difficult and time consuming, requiring specialist laboratories. As an alternative, we apply a novel approach for the diagnosis of FSHD1 utilizing single-molecule optical mapping (SMOM).
METHODS
Long DNA molecules with BssS1 enzyme marking were subjected to SMOM on the Bionano Genomics platform to determine the number of D4Z4 repeats. Southern blot and molecular combing were used to confirm the FSHD1 haplotypes.
RESULTS
In a study of a five-generation FSHD1 pedigree, SMOM correctly diagnosed the disease and normal haplotypes, identifying the founder 4qA disease allele as having 4 D4Z4 repeat units. Southern blot and molecular combing analysis confirmed the SMOM results for the 4qA disease and 4qB nondisease alleles.
CONCLUSION
Based on our findings, we propose that SMOM is a reliable and accurate technique suitable for the molecular diagnosis of FSHD1.
Topics: Genetic Testing; Humans; Muscular Dystrophy, Facioscapulohumeral; Mutation; Optical Restriction Mapping; Pedigree
PubMed: 30666819
DOI: 10.1002/mgg3.565 -
Cytogenetics and Cell Genetics 1997Quantitative DNA fiber mapping (QDFM) is a high-resolution technique for physical mapping of DNA. The method is based on hybridization of fluorescently labeled DNA...
Quantitative DNA fiber mapping (QDFM) is a high-resolution technique for physical mapping of DNA. The method is based on hybridization of fluorescently labeled DNA probes to individual DNA molecules stretched on a chemically modified glass surface. We now demonstrate and validate a rapid QDFM-based approach for the mapping of multiple restriction sites and precise localization of restriction fragments in large genomic clones. Restriction fragments of a 70-kb P1 clone (P1-70) containing the 5' region of the human apolipo-protein B gene (APOB) were subcloned and mapped along straightened P1-70 DNA molecules. Multicolor fluorescence in situ hybridization (FISH) and digital image analysis allowed us to rapidly position 29 restriction fragments, ranging in size from 0.5 kb to 8 kb, and to map 43 restriction sites. The restriction map obtained by QDFM was in excellent agreement with information obtained by RecA-assisted restriction endonuclease (RARE) cleavage, long-range PCR, and DNA sequence analyses of the P1-70 clone. These data demonstrate that QDFM is a rapid, reliable method for detailed restriction site-mapping of large DNA clones.
Topics: Apolipoproteins B; Chromosomes, Artificial, Yeast; Deoxyribonuclease EcoRI; Deoxyribonuclease HindIII; Electrophoresis; Humans; In Situ Hybridization, Fluorescence; Oligonucleotide Probes; Polymerase Chain Reaction; Rec A Recombinases; Regulatory Sequences, Nucleic Acid; Restriction Mapping; Sequence Analysis, DNA
PubMed: 9533015
DOI: 10.1159/000134685 -
Methods in Molecular Biology (Clifton,... 2016Restriction endonuclease analysis (REA) typing using HindIII enzyme is a highly discriminatory, reproducible, and consistent method of genetic typing of Clostridium...
Restriction endonuclease analysis (REA) typing using HindIII enzyme is a highly discriminatory, reproducible, and consistent method of genetic typing of Clostridium difficile (CD) isolates. REA typing analyzes CD whole cellular DNA on two levels of discrimination: REA Group designation and REA Type designation, which distinguishes specific subtypes within the REA Group. This methodology has enabled the tracking of epidemiologically significant CD strains over time and in some cases has allowed documentation of the evolution of previously rare REA Group strains that have subsequently become epidemic. The chapter details the methods used to isolate and purify CD colonies from stool samples, to obtain intact, full-length whole cellular DNA from CD isolates by use of guanidine-EDTA solution, and to analyze the HindIII-digested DNA after electrophoretic separation on agarose gels.
Topics: Bacterial Typing Techniques; Clostridioides difficile; DNA, Bacterial; DNA, Circular; Deoxyribonuclease HindIII; Electrophoresis, Agar Gel; Enterocolitis, Pseudomembranous; Feces; Humans; Prohibitins; Restriction Mapping
PubMed: 27507329
DOI: 10.1007/978-1-4939-6361-4_1 -
DNA Sequence : the Journal of DNA... Apr 2006In this study, a polymerase chain reaction (PCR) is developed to determine the restriction map without using restriction endonucleases. A 937 bp fragment of pUC 19 which...
In this study, a polymerase chain reaction (PCR) is developed to determine the restriction map without using restriction endonucleases. A 937 bp fragment of pUC 19 which contained one cut site for EcoRI and two recognition sites for PvuII was used as a model. The PCR was carried out using designed degenerate primers and the products were analyzed on 1.5% agarose gel. The number of cut sites, length of fragments and the arrangement of the fragments from 3' or 5' end of desired sequence were determined.
Topics: DNA Primers; Deoxyribonuclease EcoRI; Deoxyribonucleases, Type II Site-Specific; Plasmids; Polymerase Chain Reaction; Restriction Mapping
PubMed: 17076260
DOI: 10.1080/10425170600624834 -
Genetics Jan 1989The advent of complete genetic linkage maps consisting of codominant DNA markers [typically restriction fragment length polymorphisms (RFLPs)] has stimulated interest in...
The advent of complete genetic linkage maps consisting of codominant DNA markers [typically restriction fragment length polymorphisms (RFLPs)] has stimulated interest in the systematic genetic dissection of discrete Mendelian factors underlying quantitative traits in experimental organisms. We describe here a set of analytical methods that modify and extend the classical theory for mapping such quantitative trait loci (QTLs). These include: (i) a method of identifying promising crosses for QTL mapping by exploiting a classical formula of SEWALL WRIGHT; (ii) a method (interval mapping) for exploiting the full power of RFLP linkage maps by adapting the approach of LOD score analysis used in human genetics, to obtain accurate estimates of the genetic location and phenotypic effect of QTLs; and (iii) a method (selective genotyping) that allows a substantial reduction in the number of progeny that need to be scored with the DNA markers. In addition to the exposition of the methods, explicit graphs are provided that allow experimental geneticists to estimate, in any particular case, the number of progeny required to map QTLs underlying a quantitative trait.
Topics: Chromosome Mapping; Crosses, Genetic; Environment; Genetic Linkage; Genetic Markers; Genetic Techniques; Inbreeding; Lod Score; Mathematics; Polymorphism, Genetic; Polymorphism, Restriction Fragment Length; Recombination, Genetic; Restriction Mapping
PubMed: 2563713
DOI: 10.1093/genetics/121.1.185 -
Electrophoresis 1993This paper compares high performance capillary electrophoresis (HPCE) and conventional slab electrophoresis in mapping of four closely related plasmids with three...
This paper compares high performance capillary electrophoresis (HPCE) and conventional slab electrophoresis in mapping of four closely related plasmids with three different restriction enzymes. The plasmids express full length and truncated forms of a growth factor receptor oncogene product and were digested with HpaII, HaeIII and RsaI. The resulting oligonucleotide fragments were under 2000 base pairs in length, a size well suited to separation by HPCE with linear polyacrylamide as a sieving matrix. Plasmid mapping is an essential tool in biotechnology both for the design of an expression system and for monitoring the stability of the expression system during fermentation. HPCE can yield much higher resolution of oligonucleotides than attainable in conventional agarose gel electrophoretic procedures for plasmid mapping. In the examples described here, the HpaII digests provided the surest identification of individual plasmids in the HPCE analysis and could discriminate among all four plasmids. In conventional slab electrophoresis, however, the RsaI digests provided the best discrimination, although two of the plasmids in this system yielded essentially identical electrophoretic patterns. Hence the optimal restriction enzyme for plasmid mapping applications with HPCE may differ from that selected on the basis of conventional slab gel analysis, and the former technique can provide higher discrimination among related plasmids. The advantages of the HPCE format with respect to speed, low sample consumption and resolution are described.
Topics: Electrophoresis, Agar Gel; Electrophoresis, Polyacrylamide Gel; Plasmids; Restriction Mapping
PubMed: 8354236
DOI: 10.1002/elps.1150140178