Mechanism and Specificity of two Restriction Enzymes, CauI and CauII, that Recognize Asymmetrical DNA Sequences

Restriction Fragment Length Polymorphism (RFLP) is a powerful molecular tool that is extensively used in the molecular fingerprinting and epidemiological studies of microorganisms. In a wet-lab setting, the DNA is cut with one or more restriction enzymes and subjected to gel electrophoresis to obtain signature fragment patterns, which is utilized in the classification and identification of organisms. This wet-lab approach may not be practical when the experimental data set includes a large number of genetic sequences and a wide pool of restriction enzymes to choose from. In this study, we introduce a novel concept of Enzyme Cut Order — a biological property-based characteristic of DNA sequences which can be defined and analyzed computationally without any alignment algorithm. In this alignment-free approach, a similarity matrix is developed based on the pairwise Longest Common Subsequences (LCS) of the Enzyme Cut Orders. The choice of an ideal set of restriction enzymes used for analysis is augmented by using genetic algorithms. The results obtained from this approach using internal transcribed spacer regions of rDNA from fungi as the target sequence show that the phylogenetically-related organisms form a single cluster and successful grouping of phylogenetically close or distant organisms is dependent on the choice of restriction enzymes used in the analysis. Additionally, comparison of trees obtained with this alignment-free and the legacy method revealed highly similar tree topologies. This novel alignment-free method, which utilizes the Enzyme Cut Order and restriction enzyme profile, is a reliable alternative to local or global alignment-based classification and identification of organisms.

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Genome-Wide Identification of 5-Methylcytosine Sites in Bacterial Genomes By High-Throughput Sequencing of MspJI Restriction Fragments

10.1101/2021.02.10.430591 ◽

2021 ◽

Author(s):

Brian P. Anton ◽

Alexey Fomenkov ◽

Victoria Wu ◽

Richard J. Roberts

Keyword(s):

Single Molecule ◽

Dna Sequences ◽

High Throughput Sequencing ◽

Cost Effective ◽

Restriction Enzymes ◽

Specific Sequence ◽

Genome Wide ◽

Cost Effective Alternative ◽

Simple Column ◽

Sequencing Platforms

ABSTRACTSingle-molecule Real-Time (SMRT) sequencing can easily identify sites of N6-methyladenine and N4-methylcytosine within DNA sequences, but similar identification of 5-methylcytosine sites is not as straightforward. In prokaryotic DNA, methylation typically occurs within specific sequence contexts, or motifs, that are a property of the methyltransferases that “write” these epigenetic marks. We present here a straightforward, cost-effective alternative to both SMRT and bisulfite sequencing for the determination of prokaryotic 5-methylcytosine methylation motifs. The method, called MFRE-Seq, relies on excision and isolation of fully methylated fragments of predictable size using MspJI-Family Restriction Enzymes (MFREs), which depend on the presence of 5-methylcytosine for cleavage. We demonstrate that MFRE-Seq is compatible with both Illumina and Ion Torrent sequencing platforms and requires only a digestion step and simple column purification of size-selected digest fragments prior to standard library preparation procedures. We applied MFRE-Seq to numerous bacterial and archaeal genomic DNA preparations and successfully confirmed known motifs and identified novel ones. This method should be a useful complement to existing methodologies for studying prokaryotic methylomes and characterizing the contributing methyltransferases.

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Genome-wide identification of 5-methylcytosine sites in bacterial genomes by high-throughput sequencing of MspJI restriction fragments

PLoS ONE ◽

10.1371/journal.pone.0247541 ◽

2021 ◽

Vol 16 (5) ◽

pp. e0247541

Author(s):

Brian P. Anton ◽

Alexey Fomenkov ◽

Victoria Wu ◽

Richard J. Roberts

Keyword(s):

Single Molecule ◽

Dna Sequences ◽

High Throughput Sequencing ◽

Cost Effective ◽

Restriction Enzymes ◽

Specific Sequence ◽

Genome Wide ◽

Cost Effective Alternative ◽

Simple Column ◽

Sequencing Platforms

Single-molecule Real-Time (SMRT) sequencing can easily identify sites of N6-methyladenine and N4-methylcytosine within DNA sequences, but similar identification of 5-methylcytosine sites is not as straightforward. In prokaryotic DNA, methylation typically occurs within specific sequence contexts, or motifs, that are a property of the methyltransferases that “write” these epigenetic marks. We present here a straightforward, cost-effective alternative to both SMRT and bisulfite sequencing for the determination of prokaryotic 5-methylcytosine methylation motifs. The method, called MFRE-Seq, relies on excision and isolation of fully methylated fragments of predictable size using MspJI-Family Restriction Enzymes (MFREs), which depend on the presence of 5-methylcytosine for cleavage. We demonstrate that MFRE-Seq is compatible with both Illumina and Ion Torrent sequencing platforms and requires only a digestion step and simple column purification of size-selected digest fragments prior to standard library preparation procedures. We applied MFRE-Seq to numerous bacterial and archaeal genomic DNA preparations and successfully confirmed known motifs and identified novel ones. This method should be a useful complement to existing methodologies for studying prokaryotic methylomes and characterizing the contributing methyltransferases.

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Automata for DNA Splicing Languages with Palindromic and Non-Palindromic Restriction Enzymes using Grammars

MATEMATIKA ◽

10.11113/matematika.v35.n4.1260 ◽

2019 ◽

Vol 35 (4) ◽

pp. 1-14

Author(s):

Wan Heng Fong ◽

Nurul Izzaty Ismail ◽

Nor Haniza Sarmin

Keyword(s):

Dna Sequences ◽

Formal Language ◽

Restriction Enzymes ◽

Language Theory ◽

Dna Assembly ◽

Dna Molecules ◽

Splicing Systems ◽

Transition Graphs ◽

Palindromic Sequences ◽

Dna Splicing

In DNA splicing system, DNA molecules are cut and recombined with the presence of restriction enzymes and a ligase. The splicing system is analyzed via formal language theory where the molecules resulting from the splicing system generate a language which is called a splicing language. In nature, DNA molecules can be read in two ways; forward and backward. A sequence of string that reads the same forward and backward is known as a palindrome. Palindromic and non-palindromic sequences can also be recognized in restriction enzymes. Research on splicing languages from DNA splicing systems with palindromic and non-palindromic restriction enzymes have been done previously. This research is motivated by the problem of DNA assembly to read millions of long DNA sequences where the concepts of automata and grammars are applied in DNA splicing systems to simplify the assembly in short-read sequences. The splicing languages generated from DNA splicing systems with palindromic and nonpalindromic restriction enzymes are deduced from the grammars which are visualised as automata diagrams, and presented by transition graphs where transition labels represent the language of DNA molecules resulting from the respective DNA splicing systems.

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DNA backbone interactions impact the sequence specificity of DNA sulfur-binding domains: revelations from structural analyses

Nucleic Acids Research ◽

10.1093/nar/gkaa574 ◽

2020 ◽

Vol 48 (15) ◽

pp. 8755-8766 ◽

Cited By ~ 1

Author(s):

Hao Yu ◽

Jiayi Li ◽

Guang Liu ◽

Gong Zhao ◽

Yuli Wang ◽

...

Keyword(s):

Structural Analysis ◽

Sulfur Atom ◽

Dna Sequences ◽

Restriction Enzymes ◽

Sequence Specificity ◽

Structural Determinants ◽

Hydrogen Bonding Pattern ◽

Type Iv ◽

Phosphate Linkage ◽

Positively Charged

Abstract The sulfur atom of phosphorothioated DNA (PT-DNA) is coordinated by a surface cavity in the conserved sulfur-binding domain (SBD) of type IV restriction enzymes. However, some SBDs cannot recognize the sulfur atom in some sequence contexts. To illustrate the structural determinants for sequence specificity, we resolved the structure of SBDSpr, from endonuclease SprMcrA, in complex with DNA of GPSGCC, GPSATC and GPSAAC contexts. Structural and computational analyses explained why it binds the above PT-DNAs with an affinity in a decreasing order. The structural analysis of SBDSpr–GPSGCC and SBDSco–GPSGCC, the latter only recognizes DNA of GPSGCC, revealed that a positively charged loop above the sulfur-coordination cavity electrostatically interacts with the neighboring DNA phosphate linkage. The structural analysis indicated that the DNA–protein hydrogen bonding pattern and weak non-bonded interaction played important roles in sequence specificity of SBD protein. Exchanges of the positively-charged amino acid residues with the negatively-charged residues in the loop would enable SBDSco to extend recognization for more PT-DNA sequences, implying that type IV endonucleases can be engineered to recognize PT-DNA in novel target sequences.

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Restriction enzymes use a 24 dimensional coding space to recognize 6 base long DNA sequences

PLoS ONE ◽

10.1371/journal.pone.0222419 ◽

2019 ◽

Vol 14 (10) ◽

pp. e0222419

Author(s):

Thomas D. Schneider ◽

Vishnu Jejjala

Keyword(s):

Dna Sequences ◽

Restriction Enzymes

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Identification of Pneumococcal Serotypes by PCR–Restriction Fragment Length Polymorphism

Diagnostics ◽

10.3390/diagnostics9040196 ◽

2019 ◽

Vol 9 (4) ◽

pp. 196 ◽

Cited By ~ 1

Author(s):

García-Suárez ◽

González-Rodríguez ◽

Cima-Cabal ◽

Yuste ◽

Vazquez ◽

...

Keyword(s):

Restriction Fragment Length Polymorphism ◽

Restriction Fragment ◽

Length Polymorphism ◽

Dna Sequences ◽

Fragment Length ◽

Fragment Length Polymorphism ◽

Rflp Analysis ◽

Restriction Enzymes ◽

Pcr Rflp ◽

Restriction Fragment Length

Streptococcus pneumoniae shows more than 90 capsular serotypes that can be distinguished by their reactivity against antisera. The main objective of this work was the development of a molecular method for serotyping without the use of antisera. A computer program containing an algorithm was used to search in a database for potentially useful enzymes for Restriction Fragment Length Polymorphism-RFLP typing, in order to maximize the discrimination between different serotypes. DNA sequences of 90 serotypes for the region between dexB and aliA genes were compiled, and a computer screening of restriction enzymes was performed. The wzg–wzh–wzd–wze region and Sse9I restriction predicted unique PCR-RFLP patterns for 39 serotypes and eight serogroups. A second restriction enzyme resolved fragment specific patterns for 25 serotypes. The method was tested with 98 serotype-unknown clinical isolates. PCR-RFLP analysis deduced correct serotypes that were confirmed by Quellung reaction for 78.5% of the isolates.

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Characterization of incompatibility group HI1 plasmids from Salmonella typhi by restriction endonuclease digestion and hybridization of DNA probes for Tn3, Tn9, and Tn10

Canadian Journal of Microbiology ◽

10.1139/m85-136 ◽

1985 ◽

Vol 31 (8) ◽

pp. 721-729 ◽

Cited By ~ 7

Author(s):

Diane E. Taylor ◽

Elisa C. Brose

Keyword(s):

Dna Sequences ◽

Tetracycline Resistance ◽

Restriction Enzymes ◽

Salmonella Typhi ◽

Dna Probes ◽

Restriction Enzyme Digestion ◽

Incompatibility Group ◽

Chloramphenicol Resistance ◽

F Factor ◽

The One

Chloramphenicol resistance in Salmonella typhi is mediated by plasmids of the incompatibility group H, subgroup 1 (IncHI1). Eight IncHI1 plasmids from S. typhi strains originating in Mexico, Vietnam, Thailand, and India were examined by restriction enzyme digestion. The restriction enzymes, ApaI, XbaI, and PstI were found to be most useful for comparison of plasmid DNAs. Four plasmids from S. typhi isolated in Mexico, Vietnam, and Thailand between 1972 and 1974 had identical restriction patterns with all three enzymes. The other IncHI1 plasmids showed only minor differences. However, some significant differences were noted between these IncHI1 plasmids and the prototype IncHI1 plasmid R27, which was isolated from S. typhimurium in 1961 and for which a restriction map has been constructed. Southern transfer hybridization with a nick-translated HI1 plasmid as a probe confirmed that there is a great deal of sequence homology among the IncHI1 plasmids. DNA probes were used to locate DNA sequences for ampicillin resistance (Tn3), chloramphenicol resistance (Tn9), tetracycline resistance (Tn10), and the one-way incompatibility between IncHI1 plasmids and the F factor, a characteristic property of IncHI1 plasmids. The results demonstrate that IncHI1 plasmids isolated from S. typhi from widely different geographic sources are very similar. Comparisons between the S. typhi plasmids and R27 indicated that conserved regions of DNA were those involved in conjugative transfer.

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Discrimination of Diploid Crucifer Species Using PCR-RFLP of Chloroplast DNA

HortScience ◽

10.21273/hortsci.39.7.1575 ◽

2004 ◽

Vol 39 (7) ◽

pp. 1575-1577 ◽

Cited By ~ 6

Author(s):

Claudia Cunha ◽

Muhammet Tonguç ◽

Phillip D. Griffiths

Keyword(s):

Chloroplast Dna ◽

Dna Sequences ◽

Raphanus Sativus ◽

Fragment Length Polymorphism ◽

Diploid Species ◽

Restriction Enzymes ◽

Chain Reaction ◽

Species Identity ◽

Pcr Rflp ◽

Polymerase Chain

Chloroplast DNA (cpDNA) was used to identify polymorphisms between crucifer species using the polymerase chain reaction-random fragment-length polymorphism (PCR-RFLP) technique. Ten primer pairs based on cpDNA gene sequences were used to amplify cpDNA fragments in Brassica oleracea L., B. rapa L., B. nigra (L.) Koch, B. napus L., B. carinata Braun, B. juncea (L.) Czern, and Raphanus sativus L. accessions. Amplified DNA sequences were then digested using 11 restriction enzymes to identify polymorphisms between the 7 species. Of the 110 combinations, 38 generated polymorphisms that discriminated one or more of the species. Genotyping of these polymorphisms in 10 accessions of each of the diploid species (B. oleracea, B. nigra, B. rapa and R. sativus) did not reveal segregating polymorphisms among accessions within species, indicating that they can be used to help determine species identity. Ten accessions of each of the amphidiploids B. napus, B. carinata and B. juncea were genotyped to infer their maternal ancestry. The diploid source of cpDNA in B. carinata was B. nigra in all accessions tested and B. rapa for nine of ten B. juncea accessions tested. Two B. napus accessions amplified polymorphisms shared with B. rapa, and eight accessions produced unique polymorphisms from neither B. rapa, B. oleracea or B. nigra. The polymorphisms identified in this study can be used to help confirm identity of the diploid crucifer species for taxonomic and conservation studies.

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El uso de secuencias génicas para estudios taxonómicos

Botanical Sciences ◽

10.17129/botsci.1526 ◽

2017 ◽

pp. 137

Author(s):

Dolores González

Keyword(s):

Dna Sequences ◽

Cladistic Analysis ◽

Nuclear Genome ◽

Restriction Enzymes ◽

Dna Variation ◽

Transfer Rnas ◽

Taxonomic Research ◽

Sampling Procedures ◽

Rna Genes ◽

Molecular Characters

During the last years, the use of molecular characters for taxonomic research has increased notably. Characters most commonly used come from restriction enzymes and sequencing of genes or particular DNA regions. Sequences present potential advantages over other molecular characters. This paper describes sampling procedures to detect DNA variation through sequencing. Among procedures for DNA sequencing, enzymatic methods are of generalized application, and the tendency is toward the use of non-radioactive markers and automated sequencing. A brief introduction to the different stages of cladistic analysis is also included, especially those required for DNA sequences. A revision of genes used in systematics is provided. The most common are the nuclear, mitochondrial and chloroplast ribosomal RNA genes, and the rbcL from chloroplast. Other genes under investigation are the globins and the alcohol dehydrogenase (from the nuclear genome), the cytochrome b and the transfer RNAs (from the mitochondrial genome), and the "matK" and the "rpo" (from the chloroplast genome).

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