'DNA Strider': a 'C' program for the fast analysis of DNA and protein sequences on the Apple Macintosh family of computers

DNA Strider is a new integrated DNA and Protein sequence analysis program written with the C language for the Macintosh Plus, SE and II computers. It has been designed as an easy to learn and use program as well as a fast and efficient tool for the day-to-day sequence analysis work. The program consists of a multi-window sequence editor and of various DNA and Protein analysis functions. The editor may use 4 different types of sequences (DNA, degenerate DNA, RNA and one-letter coded protein) and can handle simultaneously 6 sequences of any type up to 32.5 kB each. Negative numbering of the bases is allowed for DNA sequences. All classical restriction and translation analysis functions are present and can be performed in any order on any open sequence or part of a sequence. The main feature of the program is that the same analysis function can be repeated several times on different sequences, thus generating multiple windows on the screen. Many graphic capabilities have been incorporated such as graphic restriction map, hydrophobicity profile and the CAI plot- codon adaptation index according to Sharp and Li. The restriction sites search uses a newly designed fast hexamer look-ahead algorithm. Typical runtime for the search of all sites with a library of 130 restriction endonucleases is 1 second per 10,000 bases. The circular graphic restriction map of the pBR322 plasmid can be therefore computed from its sequence and displayed on the Macintosh Plus screen within 2 seconds and its multiline restriction map obtained in a scrolling window within 5 seconds.     

MiCA: A Web-Based Tool for the Analysis of Microbial Communities Based on Terminal-Restriction Fragment Length Polymorphisms of 16S and 18S rRNA Genes

A web-based resource, Microbial Community Analysis (MiCA), has been developed to facilitate studies on microbial community ecology that use analyses of terminal-restriction fragment length polymorphisms (T-RFLP) of 16S and 18S rRNA genes. MiCA provides an intuitive web interface to access two specialized programs and a specially formatted database of 16S ribosomal RNA sequences. The first program performs virtual polymerase chain reaction (PCR) amplification of rRNA genes and restriction of the amplicons using primer sequences and restriction enzymes chosen by the user. This program, in silico PCR and Restriction (ISPaR), uses a binary encoding of DNA sequences to rapidly scan large numbers of sequences in databases searching for primer annealing and restriction sites while permitting the user to specify the number of mismatches in primer sequences. ISPaR supports multiple digests with up to three enzymes. The number of base pairs between the 5′ and 3′ primers and the proximal restriction sites can be reported, printed, or exported in various formats. The second program, APLAUS, infers a plausible community structure(s) based on T-RFLP data supplied by a user. APLAUS estimates the relative abundances of populations and reports a listing of phylotypes that are consistent with the empirical data. MiCA is accessible at .     

Computer-aided gene design.

A computer program, which runs on MS-DOS personal computers, is described that assists in the design of synthetic genes coding for proteins. The goal of the program is the design of a gene which (i) contains as many unique restriction sites as possible and (ii) uses a specific codon usage. The gene designed according to the criteria above is (i) suitable for 'modular mutagenesis' experiments and (ii) optimized for expression. The program 'reverse-translates' protein sequences into degenerated DNA sequences, generates a map of potential restriction sites and locates sequence positions where unique restriction sites can be accommodated. The nucleic acid sequence is then 'refined' according to a specific codon usage to remove any degeneration. Unique restriction sites, if potentially present, can be 'forced' into the degenerated nucleic acid sequence by using 'priority codes' assigned to different restriction sequences.     

Two-dimensional graphic analysis of DNA sequence homologies.

We describe a computer program designed to facilitate the pattern matching analysis of homologies between DNA sequences. It takes advantage of a two-dimensional plot in order to simplify the evaluation of significant structures inherited in the sequences. The program can be divided into three parts, i) algorithm for search of homologies, ii) two-dimensional graphic display of the result, iii) further graphic treatment to enhance significant structures. The power of the graphic display is presented by the following application of the program. We conducted a search for direct repeats in the mouse immunoglobulin kappa-chain genes. Both the five J DNA sequences and other shorter repeats were found. We also found a longer stretch of homology that could indicate the presence of duplicated DNA in the J4, J5 region.     

DNAMAT: an efficient graphic matrix sequence homology algorithm and its application to structural analysis

We present a fast algorithm to produce a graphic matrix representationof sequence homology. The algorithm is based on lexicographicalordering of fragments. It preserves most of the options of asimple naive algorithm with a significant increase in speed.This algorithm was the basis for a program, called DNAMAT, thathas been extensively tested during the last three years at theWeizmann Institute of Science and has proven to be very useful.In addition we suggest a way to extend our approach to analysea series of related DNA or RNA sequences, in order to determinecertain common structural features. The analysis is done by‘summing’ a set of dot-matrices to produce an overallmatrix that displays structural elements common to most of thesequences. We give an example of this procedure by analysingtRNA sequences. Received on June 26, 1986; accepted on September 28, 1986     

Autoscreening of Restriction Endonucleases for PCR-Restriction Fragment Length Polymorphism Identification of Fungal Species, with Pleurotus spp. as an Example

A molecular method based on PCR-restriction fragment length polymorphism (RFLP) analysis of internal transcribed spacer (ITS) ribosomal DNA sequences was designed to rapidly identify fungal species, with members of the genus Pleurotus as an example. Based on the results of phylogenetic analysis of ITS sequences from Pleurotus, a PCR-RFLP endonuclease autoscreening (PRE Auto) program was developed to screen restriction endonucleases for discriminating multiple sequences from different species. The PRE Auto program analyzes the endonuclease recognition sites and calculates the sizes of the fragments in the sequences that are imported into the program in groups according to species recognition. Every restriction endonuclease is scored through the calculation of the average coefficient for the sequence groups and the average coefficient for the sequences within a group, and then virtual electrophoresis maps for the selected restriction enzymes, based on the results of the scoring system, are displayed for the rapid determination of the candidate endonucleases. A total of 85 haplotypes representing 151 ITS sequences were used for the analysis, and 2,992 restriction endonucleases were screened to find the candidates for the identification of species. This method was verified by an experiment with 28 samples representing 12 species of Pleurotus. The results of the digestion by the restriction enzymes showed the same patterns of DNA fragments anticipated by the PRE Auto program, apart from those for four misidentified samples. ITS sequences from 14 samples (of which nine sequences were obtained in this study), including four originally misidentified samples, confirmed the species identities revealed by the PCR-RFLP analysis. The method developed here can be used for the identification of species of other living microorganisms.     

A general DNA analysis program for the Hewlett-Packard Model 86/87 microcomputer.

A program is described to perform general DNA sequence analysis on the Hewlett-Packard Model 86/87 microcomputer operating on 128 K of RAM. The following analytical procedures can be performed: 1. display of the sequence, in whole or part, or its complement; 2. search for specified sequences e.g. restriction sites, and in the case of the latter give fragment sizes; 3. perform a comprehensive search for all known restriction enzyme sites; 4. map sites graphically; 5. perform editing functions; 6. base frequency analysis; 7. search for repeated sequences; 8. search for open reading frames or translate into the amino acid sequence and analyse for basic and acidic amino acids, hydrophobicity, and codon usage. Two sequences, or parts thereof, can be merged in various orientations to mimic recombination strategies, or can be compared for homologies. The program is written in HP BASIC and is designed principally as a tool for the laboratory investigator manipulating a defined set of vectors and recombinant DNA constructs.     

Hypervariable single and multi-locus DNA polymorphisms for genetic typing of non-human primates

The series of hypervariable, “minisatellite” loci characterized byJeffreys and coworkers in the human myoglobin gene have proved to be DNA sequences highly conserved throughout the eukaryotic genome, and hence the methodology developed for human DNA “fingerprinting” has found immediate application in an ever expanding number of species. Primatologists have not been slow to profit from a method which predicts individual recognition to a very high degree of probability, and initial studies have focused on paternity allocation (rather than paternity exclusion, as designated by the classical biochemical markers), adaptive aspects of socio-sexual behaviour patterns and mating systems. A number of probes with sequences corresponding to the common minisatellite core sequences have been used for probing genomic DNA, and synthetic, G-rich oligonucleotides (15 – 37 bases), corresponding to the core sequence of the minisatellite repeat unit, or simply di-, tri-, or tetranucleotide repeats, appear to be equally discriminatory. The multiple banding patterns produced on hybridization of these probes to restriction enzyme digests of DNA provide an advantage in that the probability of two unrelated individuals sharing the same banding pattern will be low. However, the uncertainty of linkage of the multiple loci identified precludes genotyping and population genetic analyses based on allele frequencies. In contrast, single locus analysis allows DNA typing using variable number tandem repeat (VNTR) or restriction fragment length (RFLP) DNA polymorphisms, and the merits and drawbacks relative to DNA fingerprinting are discussed. For the behavioural primatologists dealing with defined, accessible troops of primates, the value of multilocus DNA fingerprinting, in terms of established methodology and availability of probes applicable to species as phylogenetically wide-ranging as apes and prosimians, may well outweigh the loss of genotypic and population structure data.     

A plasmid rescue technique for the recovery of plant DNA disrupted by T-DNA insertion

Arabidopsis mutants generated by insertion of the T-DNA from Ti plasmid 3850∶1003 serve as a starting point for the isolation of novel genes. The disrupted plant DNA can be recovered using a plasmid rescue technique utilizing high efficiency electroporation. Rescued plasmids are resistant to ampicillin and contain an origin of replication from pBR322. Plasmids generated from either the left or right border of the T-DNA that carry flanking DNA sequences can be identified by analyzing the products of restriction enzyme digests on agarose gels. The plasmids with flanking sequences can then serve as a starting point for cloning plant sequences that share homology to the DNA at the point of T-DNA insertion.     

Spreadsheet-based program for alignment of overlapping DNA sequences.

Molecular biology laboratories frequently face the challenge of aligning small overlapping DNA sequences derived from a long DNA segment. Here, we present a short program that can be used to adapt Excel spreadsheets as a tool for aligning DNA sequences, regardless of their orientation. The program runs on any Windows or Macintosh operating system computer with Excel 97 or Excel 98. The program is available for use as an Excel file, which can be downloaded from the BioTechniques Web site. Upon execution, the program opens a specially designed customized workbook and is capable of identifying overlapping regions between two sequence fragments and displaying the sequence alignment. It also performs a number of specialized functions such as recognition of restriction enzyme cutting sites and CpG island mapping without costly specialized software.     

In Silico Analysis of the Restriction Fragment Length Distribution in the Human Genome

The Restriction On Computer (ROC) program (freely available at http://www.mcb.harvard.edu/ gilbert/ROC) was developed and used to analyze the restriction fragment length distribution in the human genome. In contrast to other programs searching for restriction sites, ROC simultaneously analyzes several long nucleotide sequences, such as the entire genomes, and in essence simulates electrophoretic analysis of DNA restriction fragments. In addition, this program extracts and analyzes DNA repeats that account for peaks in the restriction fragment length distribution. The ROC analysis data are consistent with the experimental data obtained via in vitro restriction enzyme analysis (DNA taxonoprint). A difference between the in vitro and in silico results is explained by underrepresentation of tandem DNA repeats in genomic databases. The ROC analysis of individual genome fragments elucidated the nature of several DNA markers, which were earlier revealed by DNA taxonoprint, and showed that L1 and Alurepeats are nonrandomly distributed in various chromosomes. Another advantage is that the ROC procedure makes it possible to analyze the nonrandom character of a genomic distribution of short DNA sequences. The ROC analysis showed that a low poly(G) frequency is characteristic of the entire human genome, rather than of only coding sequences. The method was proposed for a more complex in silico analysis of the genome. For instance, it is possible to simulate DNA restriction together with blot hybridization and then to analyze the nature of markers revealed.     

A computer assisted method for the determination of restriction enzyme recognifion sites.

A computer program has been developed which aids in the determination of restriction enzyme recognition sequences. This is achieved by cleaving DNAs of known sequence with a restriction endonuclease and comparing the fragmentation pattern with a computer-generated set of patterns. The feasibility of this approach has been tested using fragmentation patterns of 0X174 DNA produced by enzymes of both known and unknown specificity. Recognition sequences are predicted for two restriction endonucleases (BbvI and SfaNI) using this method. In addition, recognition sequences are predicted for two other new enzymes (PvuI and MstI) using another computer-assisted method.     

An improved FORTRAN 77 recombinant DNA database management system with graphic extensions in GKS

We have improved an existing clone database management systemwritten in FORTRAN 77 and adapted it to our software environment.Improvements are that the database can be interrogated for anytype of information, not just keywords. Also, recombinant DNAconstructions can be represented in a simplified ‘shorthand’,whereafter a program assembles the full nucleotide sequencefrom the contributing fragments, which may be obtained fromnucleotide sequence databases. Another improvement is the replacementof the database manager by programs, running in batch to maintainthe databank and verify its consistency automatically. Finally,graphic extensions are written in Graphical Kernel System, todraw linear and circular restriction maps of recombinants. Besidesrestriction sites, recombinant features can be presented fromthe feature lines of recombinant database entries, or from thefeature tables of nucleotide databases. The clone database managementsystem is fully integrated into the sequence analysis softwarepackage from the Pasteur Institute, Paris, and is made accessiblethrough the same menu. As a result, recombinant DNA sequencescan directly be analysed by the sequence analysis programs. Received on March 17, 1986; accepted on June 16, 1986     

A peptide to DNA conversion program.

A modification and extension of the computer program REVCUT (Blumenthal et al, Nucl.Acids Res. 10, 91-101 (1982) is described. The new program searches for restriction endonuclease recognition sites that are not coding DNA sequences of a protein of known aminoacid sequence using bit patterns. The modifications make the program more accurate and extend the range of the restriction endonucleases.     

SILMUT: a computer program for the identification of regions suitable for silent mutagenesis to introduce restriction enzyme recognition sequences.

We describe a set of IBM-compatible computer programs designed to selectively identify the potential sites for silent mutagenesis within a target DNA sequence. This program is based on a novel strategy of identifying amino acid motifs compatible with each restriction site (BioTechniques 12:382-384, 1991). The programs can be used to identify the suitability for the introduction of any 6-base nucleic acid sequences, such as restriction enzyme sites in cassette mutagenesis strategies. The Table program generates a table of multiple amino acid motifs for each restriction enzyme, obtained by translating each unique recognition sequence in all three reading frames. The Silmut program, which utilizes the features of Table, will further identify the presence of a match between any amino acid motif of each restriction enzyme and the input target sequence. Minor manipulations of the data base files will enable the individual researcher to identify the potential for introduction of any 6-base sequences by silent mutagenesis.     

Cloning genomic sequences using long-range PCR

Protocols are presented for preparing DNA from a genomic library in λ phage and for synthesizing genomic fragments using PCR with nested vector- and gene-specific primers and linker-primers. Library DNA, isolated fromE. coli liquid lysates by a simple protocol, is used as template in PCR following a commercial protocol. The method produces library DNA sufficient for several hundred PCRs, incorporates nested primers to reduce nonspecific product formation, and enables the synthesis of linker-containing DNA fragments containing selected restriction sites to simplify subsequent cloning. The isolation of 5′ upstream sequences of three different arabidopsis genes by this methodod is described.     

Flotation as a tool for indirect DNA extraction from soil

Nowadays, soil diversity is accessed at molecular level by the total DNA extraction of a given habitat. However, high DNA yields and purity are difficult to achieve due to the co-extraction of enzyme-inhibitory substances that inhibit downstream applications, such as PCR, restriction enzyme digestion, and DNA ligation. Therefore, there is a need for further development of sample preparation methods that efficiently can result in pure DNA with satisfactory yield. In this study, the buoyant densities of soil microorganisms were utilized to design a sample preparation protocol where microbial cells could be separated from the soil matrix and enzyme-inhibitory substances by flotation. A discontinuous density gradient was designed using a colloidal solution of non-toxic silanised silica particles (BactXtractor). The method proved to be an efficient alternative to direct extraction protocols where cell lysis is performed in the presence of soil particles. The environmental DNA extracted after flotation had high molecular weight and comparable yield as when using available commercial kits (3.5 μg DNA/g soil), and neither PCR nor restriction enzyme digestion of DNA were inhibited. Furthermore, specific primers enabled recovery of both prokaryotic and eukaryotic sequences.     

Probing of unusual DNA structures in topologically constrained form V DNA: use of restriction enzymes as structural probe.

The ability of DNA sequences to adopt unusual structures under the superhelical torsional stress has been studied. Sequences that are forced to adopt unusual conformation in topologically constrained pBR322 form V DNA (Lk = 0) were mapped using restriction enzymes as probes. Restriction enzymes such as BamHI, PstI, AvaI and HindIII could not cleave their recognition sequences. The removal of topological constraint relieved this inhibition. The influence of neighbouring sequences on the ability of a given sequence to adopt unusual DNA structure, presumably left handed Z conformation, was studied through single hit analysis. Using multiple cut restriction enzymes such as NarI and FspI, it could be shown that under identical topological strain, the extent of structural alteration is greatly influenced by the neighbouring sequences. In the light of the variety of sequences and locations that could be mapped to adopt non-B conformation in pBR322 form V DNA, restriction enzymes appear as potential structural probes for natural DNA sequences.     

PlasmaDNA: a free, cross-platform plasmid manipulation program for molecular biology laboratories

Background

Most molecular biology experiments, and the techniques associated with this field of study, involve a great deal of engineering in the form of molecular cloning. Like all forms of engineering, perfect information about the starting material is crucial for successful completion of design and strategies.

Results

We have generated a program that allows complete in silico simulation of the cloning experiment. Starting with a primary DNA sequence, PlasmaDNA looks for restriction sites, open reading frames, primer annealing sequences, and various common domains. The databases are easily expandable by the user to fit his most common cloning needs. PlasmaDNA can manage and graphically represent multiple sequences at the same time, and keeps in memory the overhangs at the end of the sequences if any. This means that it is possible to virtually digest fragments, to add the digestion products to the project, and to ligate together fragments with compatible ends to generate the new sequences. Polymerase Chain Reaction (PCR) fragments can also be virtually generated using the primer database, automatically adding to the fragments any 5' extra sequences present in the primers.

Conclusion

PlasmaDNA is a program available both on Windows and Apple operating systems, designed to facilitate molecular cloning experiments by building a visual map of the DNA. It then allows the complete planning and simulation of the cloning experiment. It also automatically updates the new sequences generated in the process, which is an important help in practice. The capacity to maintain multiple sequences in the same file can also be used to archive the various steps and strategies involved in the cloning of each construct. The program is freely available for download without charge or restriction.