IBM microcomputer programs that analyze DNA sequences for tRNA genes

A set of four computer programs that search DNA sequence datafiles for transfer RNA genes have been written in IBM (Microsoft)BASIC for the IBM personal computer. These programs locate andplot predicted secondary structures of tRNA genes in the cloverleafconformation. The set of programs are applicable to eukaryotictRNA genes, including those containing intervening sequences,and to prokaryotic and mitochondrial tRNA genes. In addition,two of the programs search up to 150 residues downstream oftRNA gene sequences for possible eukaryotic RNA polymerase IIItermination sites comprised of at least four consecutive T residues.Molecular biologists studying a variety of gene sequences andflanking regions can use these programs to search for the additionalpresence of tRNA genes. Furthermore, investigators studyingtRNA gene structure-to-function relationships would not needto do extensive restriction mapping to locate tRNA gene sequenceswithin their cloned DNA fragments. Received on October 29, 1985; accepted on January 28, 1986     

Microcomputer programs for DNA sequence analysis.

Computer programs are described which allow (a) analysis of DNA sequences to be performed on a laboratory microcomputer or (b) transfer of DNA sequences between a laboratory microcomputer and another computer system, such as a DNA library. The sequence analysis programs are interactive, do not require prior experience with computers and in many other respects resemble programs which have been written for larger computer systems (1-7). The user enters sequence data into a text file, accesses this file with the programs, and is then able to (a) search for restriction enzyme sites or other specified sequences, (b) translate in one or more reading frames in one or both directions in order to find open reading frames, or (c) determine codon usage in the sequence in one or more given reading frames. The results are given in table format and a restriction map is generated. The modem program permits collection of large amounts of data from a sequence library into a permanent file on the microcomputer disc system, or transfer of laboratory data in the reverse direction to a remote computer system.     

Fast analysis of DNA and protein sequence on Apple IIe: restriction sites search, alignment of short sequence and dot matrix analysis.

A fast restriction sites search algorithm using a quadruplet look-ahead feature has been written in 6502 assembly language code. The search time, tested on the sequence of pBR322, is 4.1 s/kilobase using a restriction site library including 112 specificities corresponding to a total site length of over 700 bases. The search for a short sequence (less than 36 bases) within a longer one (up to 9999 bases) with a given number of mismatches or gaps allowed has also been written in assembly language. Typical run time for the search of a 12 base sequence with 1, 2 or 3 gaps allowed are 6.2, 9.4 or 13.6 s/kilobase, respectively. The dot matrix analysis needs 7.5 minutes per square kilobase when using a stringency of 15 matched bases out of 25. A 7/21 matrix of two 500 amino acid proteins is obtained in 3 minutes. These three routines are included in DPSA, a general package of programs allowing manipulation and analysis of DNA and protein sequences.     

A program package applicable to the detection of overlaps between restriction maps.

A computer program package for the storage, change, and comparison of restriction maps is described. The programs are intended to detect overlaps between relatively short (about 10-40 kb; abbreviations ref.2) maps and to merge the overlapping fragments into large restriction maps. They run on a 16-bit-microcomputer with limited memory and addressing capability. Due to the restricted reliability of restriction maps compared with DNA sequence data a particular storage method was used. The source code of the programs is freely available (+).     

A set of Macintosh computer programs for the design and analysis of synthetic genes

Computer programs that can be used for the design of syntheticgenes and that are run on an Apple Macintosh computer are described.These programs determine nucleic acid sequences encoding aminoacid sequences. They select DNA sequences based on codon usageas specified by the user, and determine the placement of basechanges that can be used to create restriction enzyme siteswithout altering the amino acid sequence. A new algorithm forfinding restriction sites by translating the restriction endonucleasetarget sequence in all three reading frames and then searchingthe given peptide or protein amino acid sequence with theseshort restriction enzyme peptide sequences is described. Examplesare given for the creation of synthetic DNA sequences for thebovine prethrombin-2 and ribonuclease A genes Received on October 18, 1988; accepted on December 9, 1988     

Comprehensive restriction enzyme lists to update any DNA sequence computer program

Restriction enzyme lists are presented for the practical working geneticist to update any DNA computer program. These lists combine formerly scattered information and contain all presently known restriction enzymes with a unique recognition sequence, a cut site, or methylation (in)sensitivity. The lists are in the shortest possible form to also be functional with small DNA computer programs, and will produce clear restriction maps without any redundancy or loss of information. The lists discern between commercial and noncommercial enzymes, and prototype enzymes and different isoschizomers are cross-referenced. Differences in general methylation sensitivities and (in)sensitivities against Dam and Dcm methylases of Escherichia coli are indicated. Commercial methylases and intron-encoded endonucleases are included. An address list is presented to contact commercial suppliers. The lists are constantly updated and available in electronic form as pure US ASCII files, and in formats for the DNA computer programs DNA-Strider for Apple Macintosh, and DNAsis for IBM personal computers or compatibles via e-mail from the internet address: netservembl-heidelberg.de by sending only the message help relibrary.     

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.     

'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.     

The design of synthetic genes

Computer programs are described that aid in the design of synthetic genes coding for proteins that are targets of a research program in site directed mutagenesis. These programs "reverse-translate" protein sequences into general nucleic acid sequences (those where codons have not yet been selected), map restriction sites into general DNA sequences, identify points in the synthetic gene where unique restriction sites can be introduced, and assist in the design of genes coding for hybrids and evolutionary intermediates between homologous proteins. Application of these programs therefore facilitates the use of modular mutagenesis to create variants of proteins, and the implementation of evolutionary guidance as a strategy for selecting mutants.     

A model for restriction fragment length distributions.

We develop here a model for restriction fragment length distributions based on DNA dimer frequencies in humans. Mean fragment lengths are computed for known restriction enzymes. This model is tested using data from the hybridization of a series of arbitrary single-copy DNA probes screened with a set of restriction enzymes. The fit to the model appears good. We apply the model to the problem of how much DNA is scanned by a set of enzymes. This result is then further applied to optimizing the search for insertion/deletion DNA-polymorphisms.     

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.     

A search for restriction fragment length polymorphism on the human Y chromosome

Summary A systematic search for restriction fragment length polymorphisms (RFLPs) on the human Y chromosome was performed. DNA samples from 16–34 individuals were screened with five restriction enzymes and 12 Y-chromosomal probes, 3 of which detect lowly repetitive sequences and 9 of which are apparently single copy in genomic DNA. None of the single-copy probes revealed any variation. The repetitive sequence probe p21A1 (DYZ?) revealed a TaqI RFLP with q = 0.05. The frequency of fixed point mutations in Y-chromosomal DNA outside the pseudoautosomal region is probably less than 1 in 18000 bp.     

In silico analysis of the restriction fragments 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 (taxonomic printing). 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 taxonomic printing, and showed that L1 and Alu repeats 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.     

Alignment of Escherichia coli K12 DNA sequences to a genomic restriction map.

We use the extensive published information describing the genome of Escherichia coli and new restriction map alignment software to align DNA sequence, genetic, and physical maps. Restriction map alignment software is used which considers restriction maps as strings analogous to DNA or protein sequences except that two values, enzyme name and DNA base address, are associated with each position on the string. The resulting alignments reveal a nearly linear relationship between the physical and genetic maps of the E. coli chromosome. Physical map comparisons with the 1976, 1980, and 1983 genetic maps demonstrate a better fit with the more recent maps. The results of these alignments are genomic kilobase coordinates, orientation and rank of the alignment that best fits the genetic data. A statistical measure based on extreme value distribution is applied to the alignments. Additional computer analyses allow us to estimate the accuracy of the published E. coli genomic restriction map, simulate rearrangements of the bacterial chromosome, and search for repetitive DNA. The procedures we used are general enough to be applicable to other genome mapping projects.     

Restriction enzyme cutting site distribution regularity for DNA looping technology

The restriction enzyme cutting site distribution regularity and looping conditions were studied systematically. We obtained the restriction enzyme cutting site distributions of 13 commonly used restriction enzymes in 5 model organism genomes through two novel self-compiled software programs. All of the average distances between two adjacent restriction sites fell sharply with increasing statistic intervals, and most fragments were 0–499 bp. A shorter DNA fragment resulted in a lower looping rate, which was also directly proportional to the DNA concentration. When the length was more than 500 bp, the concentration did not affect the looping rate. Therefore, the best known fragment length was longer than 500 bp, and did not contain the restriction enzyme cutting sites which would be used for digestion. In order to make the looping efficiencies reach nearly 100%, 4–5 single cohesive end systems were recommended to digest the genome separately.     

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.     

In silico analysis of complete bacterial genomes: PCR, AFLP-PCR and endonuclease restriction

We have developed a website, www.in-silico.com, which runs a software program that performs three basic tasks in completely sequenced bacterial genomes by in silico analysis: PCR amplification, amplified fragment length polymorphism (AFLP-PCR) and endonuclease restriction. For PCR, after selection of the genome and introduction of primers, fragment size, DNA sequence and corresponding open reading frame (ORF) identity of the resulting PCR product is computed. Plasmids of sequenced species may be included in the analysis. Theoretical AFLP-PCR analyzes similar parameters, and includes a suggestion tool providing a list of commercial restriction enzyme pairs yielding up to 50 amplicons in the selected genome. Endonuclease restriction analysis of complete genomes and plasmids calculates the number of restriction sites for endonucleases in a given genome. If the number of fragments is 50 or fewer, pulsed field gel electrophoresis image and restriction maps are illustrated. Other tools that have been included in this site are ORF search by name and DNA to protein translation as well as restriction digestion of user-defined DNA sequences. AVAILABILITY: This is a new molecular biology resource freely available over the Internet at http://www.in-silico.com     

Sequence Variation in the Ribosomal DNA Internal Transcribed Spacer of Tridacna crocea

DNA-based genetic markers are needed to augment existing allozyme markers in the assessment of genetic diversity of wild giant clam populations. The dearth of polymorphic mitochondrial DNA regions amplified from known universal polymerase chain reaction (PCR) primers has led us to search other regions of the genome for viable sources of DNA polymorphism. We have designed tridacnid-specific PCR primers for the amplification of internal transcribed spacer regions. Sequences of the first internal transcribed spacer segment (ITS-1) revealed very high polymorphism, showing 29% variation arising from base substitutions alone. Preliminary restriction analysis of the ITS regions using 8 restriction enzymes revealed cryptic changes in the DNA sequence. These mutations are promising as marker tools for differentiating geographically separated populations. Such variation in the ITS region can possibly be used for population genetic analysis. Received February 1, 2000; accepted May 8, 2000.     

C.U.R.R.F. (Codon Usage regarding Restriction Finder): A Free Java?-Based Tool to Detect Potential Restriction Sites in Both Coding and Non-Coding DNA Sequences

The synthesis of complete genes is becoming a more and more popular approach in heterologous gene expression. Reasons for this are the decreasing prices and the numerous advantages in comparison to classic molecular cloning methods. Two of these advantages are the possibility to adapt the codon usage to the host organism and the option to introduce restriction enzyme target sites of choice. C.U.R.R.F. (Codon Usage regarding Restriction Finder) is a free Java(?)-based software program which is able to detect possible restriction sites in both coding and non-coding DNA sequences by introducing multiple silent or non-silent mutations, respectively. The deviation of an alternative sequence containing a desired restriction motive from the sequence with the optimal codon usage is considered during the search of potential restriction sites in coding DNA and mRNA sequences as well as protein sequences. C.U.R.R.F is available at http://www.zvm.tu-dresden.de/die_tu_dresden/fakultaeten/fakultaet_mathematik_und_naturwissenschaften/fachrichtung_biologie/mikrobiologie/allgemeine_mikrobiologie/currf .     

Word-processor macro for restriction endonuclease analysis

This paper describes a Microsoft Word 97 macro designed for restriction endonuclease analysis. Selected DNA fragments in the active Word document can be analyzed through a dynamic dialog box that formats the enzyme restriction lists for further analysis. The results can be obtained in a new Word document with the name of the enzymes, number of cuts and positions. This macro has several advantages: the results can be printed in a format suitable for record keeping, no additional programs are required and it is simple to use.