Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension

Gene splicing by overlap extension is a new approach for recombining DNA molecules at precise junctions irrespective of nucleotide sequences at the recombination site and without the use of restriction endonucleases or ligase. Fragments from the genes that are to be recombined are generated in separate polymerase chain reactions (PCRs). The primers are designed so that the ends of the products contain complementary sequences. When these PCR products are mixed, denatured, and reannealed, the strands having the matching sequences at their 3' ends overlap and act as primers for each other. Extension of this overlap by DNA polymerase produces a molecule in which the original sequences are 'spliced' together. This technique is used to construct a gene encoding a mosaic fusion protein comprised of parts of two different class-I major histocompatibility genes. This simple and widely applicable approach has significant advantages over standard recombinant DNA techniques.     

Characterization of the genome of the Rhodococcus rhodochrous bacteriophage NJL.

Temperate bacteriophage NJL of Rhodococcus rhodochrous has a 49-kb linear double-stranded DNA with cohesive ends (cos). NJL DNA has unique target sites for HindIII and SspI, two target sites each for NheI and ScaI, and no cleavage site for AxyI, DraI, EcoRI, SacI, and SphI. The single-stranded regions of cos ends were ligated to each other with T4 DNA ligase, removed with mung bean nuclease, or blunted with the Klenow large fragment of DNA polymerase I; then the sequences of the cos ends were determined. Comparison of these sequences revealed that the single-stranded regions are complementary and 18 bases long and protrude at the 3' ends; they have the following sequences: 5'-TTGGCACCGTGGGAGGAG-3' and 3'-AACCGTGGCAC CCTCCTC-5'. A physical map of NJL was constructed by a cos mapping method based on information about the structure of the cohesive ends and multiple digestions with restriction endonucleases.     

In silico simulation of fingerprinting techniques based on double endonuclease digestion of genomic DNA

We have developed an online generic tool for simulation of fingerprinting techniques based on the double endonuclease digestion of DNA. This tool allows modelling and modifications of already existing techniques, as well as new theoretical approaches not yet tried in the lab. It allows the use of any combination of recognition patterns and discrimination of end types yielded by restriction with non palindromic recognition sizes. Re-creation of experimental conditions in silico saves time and reduces laboratory costs. This tool allows simulation of Amplified Fragment Length Polymorphism (AFLP-PCR), Subtracted Restriction Fingerprinting (SRF), and additional novel fingerprinting techniques. Simulation may be performed against custom sequences uploaded to the server, or against all sequenced bacterial genomes. Different endonuclease types may be selected from a list, or a recognition sequence may be introduced in the form. After double digestion of DNA, four fragment types are yielded, and the program allows their customised selection. Selective nucleotides may be used in the experiment. Scripts for specific simulation of AFLP-PCR and SRF techniques are available, and both include a suggestion tool for the selection of endonucleases. This is the first program available for the simulation of SRF fingerprinting. Availability: This free online tool is available at http://www.in-silico.com/DDF/.     

Cloning of random-sequence oligodeoxynucleotides

Methods are described for cloning random or highly degenerate nucleotide (nt) sequences. The procedures use synthetically derived mixtures of oligodeoxynucleotides (oligos) whose heterogeneous central portions are bounded at their 5' and 3' ends by sequences recognized by restriction endonucleases. Oligo collections of defined length and nt composition are synthesized by utilizing appropriate concentrations of all four nucleotide precursors during each addition step for the central region. Single-stranded oligos with appropriate 5' and 3' ends can be ligated directly, although inefficiently, into double-stranded (ds) DNA molecules with complementary 5' and 3' extensions produced by restriction endonuclease cleavage. A more general and efficient method is to convert the oligo into a ds form by incubating it with the Klenow (large) fragment of Escherichia coli DNA polymerase I. If the 3' ends are palindromic, two oligo molecules will serve as mutual primers for polymerization. The resulting products are ds molecules containing two oligo units separated by the original 3' restriction site and bounded at each end by the original 5' restriction site. After appropriate restriction endonuclease cleavage, oligo units can be cloned by standard procedures. Analysis of 26 recombinant M13 phages indicates that the nt sequences of the cloned oligos are in good accord with what was expected on a random basis.     

Intramolecular transposition by Tn10

Transposon Tn10 promotes the formation of a circular product containing only transposon sequences. We show that these circles result from an intramolecular transposition reaction in which all of the strand cleavage and ligation events have occurred but newly created transposon/target junctions have not undergone repair. The unligated strand termini at these junctions are those expected according to a simple model in which the target DNA is cleaved by a pair of staggered nicks 9 bp apart, transposon sequences are separated from flanking donor DNA by cleavage at the terminal nucleotides on both strands (at both ends) of the element, and 3' transposon strand ends are ligated to 5' target strand ends. The stability of the unligated junctions suggests that they are protected from cellular processing by transposase and/or host proteins. We propose that the nonreplicative nature of Tn10 transposition is determined by the efficiency with which the nontransferred transposon strand is separated from flanking donor DNA and by the nature of the protein-DNA complexes present at the strand transfer junctions.     

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.     

A program for the graphic representation and manipulation of DNA sequences

We have developed a program for the graphic representation andmanipulation of DNA sequences. The program (named CARTE fromthe French for ‘map’) is intended as a tool in theplanning and analysis of recombinant DNA experiments. DNA sequencesare represented as standard restriction maps, using any desiredcombination of restriction enzymes. Features of interest, suchas promoters or coding sequences, can be highlighted. The sequencecan be manipulated to mimic cloning, using deletions, insertionsor replacements at specified sites. This process is facilitatedby the simultaneous display of a graphic map of the entire sequence,a detailed picture of the work in progress, and a menu of functions. Received on November 17, 1986; accepted on March 12, 1987     

An access interface for the MS-DOS diskette format of GenBank(R), a gene sequence database

An interface program has been developed for users of MS-DOScomputers and the GenBank(R) gene sequence files in their disketteformat. With the program a user is able to produce keyword,author and entry name listings of GenBank items or to selectGenBank sequences for viewing, printing or decoding. The decodeoption uncompresses sequence data and yields a character filewhich has the format used on GenBank magnetic tapes. Programoptions are chosen by selecting items from command menus. Whilethe program is designed primarily for hard disk operation, italso allows users of diskette-based computers to work with GenBankfiles. Received on July 15, 1987; accepted on July 15, 1987     

A flexible method to align large numbers of biological sequences

Summary A method for the alignment of two or more biological sequences is described. The method is a direct extension of the method of Taylor (1987) incorporating a consensus sequence approach and allows considerable freedom in the control of the clustering of the sequences. At one extreme this is equivalent to the earlier method (Taylor 1987), whereas at the other, the clustering approaches the binary method of Feng and Doolittle (1987). Such freedom allows the program to be adapted to particular problems, which has the important advantage of resulting in considerable savings in computer time, allowing very large problems to be tackled. Besides a detailed analysis of the alignment of the cytochrome c superfamily, the clustering and alignment of the PIR sequence data bank (3500 sequences approx.) is described.     

ENDO-Pore: high-throughput linked-end mapping of single DNA cleavage events using nanopore sequencing

Mapping the precise position of DNA cleavage events plays a key role in determining the mechanism and function of endonucleases. ENDO-Pore is a high-throughput nanopore-based method that allows the time resolved mapping single molecule DNA cleavage events in vitro. Following linearisation of a circular DNA substrate by the endonuclease, a resistance cassette is ligated recording the position of the cleavage event. A library of single cleavage events is constructed and subjected to rolling circle amplification to generate concatemers. These are sequenced and used to produce accurate consensus sequences. To identify the cleavage site(s), we developed CSI (Cleavage Site Investigator). CSI recognizes the ends of the cassette ligated into the cleaved substrate and triangulates the position of the dsDNA break. We firstly benchmarked ENDO-Pore using Type II restriction endonucleases. Secondly, we analysed the effect of crRNA length on the cleavage pattern of CRISPR Cas12a. Finally, we mapped the time-resolved DNA cleavage by the Type ISP restriction endonuclease LlaGI that introduces random double-strand breaks into its DNA substrates.     

Oligonucleotide capture during end joining in mammalian cells.

Extra nucleotides (termed filler DNA) are found at about 10% of the junctions of the genetic rearrangements that arise by illegitimate recombination in mammalian cells. Such filler DNAs could arise by the joining of oligonucleotide fragments to broken ends prior to end joining. We tested this possibility by microinjecting mixtures of defined oligonucleotides with SV40 genomes that were linearized in the intron for T antigen, a site where incorporation of extra nucleotides does not impair viability. Using an injection ratio of 1000 oligonucleotides per DNA end, we screened viable genomes for incorporation of single-stranded and double-stranded oligonucleotides with varying degrees of complementarity to the ends of the linear SV40 molecules. Genomes from 510 independent plaques were screened by restriction digestion to identify those that had picked up a restriction site unique to the injected oligonucleotides. Double-stranded oligonucleotides that were fully complementary to the SV40 ends were readily incorporated, but uptake of the other oligonucleotides was not detected by restriction analysis. Nucleotide sequences of junctions from 12 genomes derived from co-injection of noncomplementary oligonucleotides revealed two with filler DNA, but neither could be assigned unambiguously to the injected oligonucleotides.     

A simple ligation-based method to increase the information density in sequencing reactions used to deconvolute nucleic acid selections

Herein, a method is described to increase the information density of sequencing experiments used to deconvolute nucleic acid selections. The method is facile and should be applicable to any selection experiment. A critical feature of this method is the use of biotinylated primers to amplify and encode a BamHI restriction site on both ends of a PCR product. After amplification, the PCR reaction is captured onto streptavidin resin, washed, and digested directly on the resin. Resin-based digestion affords clean product that is devoid of partially digested products and unincorporated PCR primers. The product's complementary ends are annealed and ligated together with T4 DNA ligase. Analysis of ligation products shows formation of concatemers of different length and little detectable monomer. Sequencing results produced data that routinely contained three to four copies of the library. This method allows for more efficient formulation of structure-activity relationships since multiple active sequences are identified from a single clone.     

High-efficiency DNA ligation for clamp attachment without polymerase chain reaction

We coupled ligation with mass action to achieve high-efficiency clamp attachment without polymerase chain reaction (PCR). Using a 10-fold molar excess of a GC-rich clamp of synthesized and hybridized oligonucleotides, we achieved the maximum clamp-ligation efficiency in which the clamp was ligated to >95% of 10(10)-10(12) restriction ends of a PCR-amplified fragment. The maximum efficiency was confirmed by ligating the clamp to 10(11)-10(12) restriction ends of human genomic DNA. Our approach can be added to a constant denaturant capillary electrophoresis (CDCE)-based method of analyzing rare point mutants at fractions as low as 10(-6); such mutants appear as small copy numbers in the initial samples. This CDCE-based method alone is applicable to only those DNA sequences juxtaposed with an internally occurring clamp of a higher melting temperature in genomic DNA. Since such sequences represent 9% of the human genome, the addition of clamp ligation significantly increases the scanning range for the human genome without reducing the initial mutant copy numbers. Furthermore, clamp ligation/attachment without PCR prevents PCR-created mutants from interfering with rare mutational analysis. In addition to those applications seeking high-efficiency DNA ligation, our approach can be generally applied to ligation of restriction ends.     

Mechanistic constraints on diversity in human V(D)J recombination.

We have analyzed a large collection of coding junctions generated in human cells. From this analysis, we infer the following about nucleotide processing at coding joints in human cells. First, the pattern of nucleotide loss from coding ends is influenced by the base composition of the coding end sequences. AT-rich sequences suffer greater loss than do GC-rich sequences. Second, inverted repeats can occur at ends that have undergone nucleolytic processing. Previously, inverted repeats (P nucleotides) have been noted only at coding ends that have not undergone nucleolytic processing, this observation being the basis for a model in which a hairpin intermediate is formed at the coding ends early in the reaction. Here, inverted repeats at processed coding ends were present at approximately twice the number of junctions as P nucleotide additions. Terminal deoxynucleotidyl transferase (TdT) is required for the appearance of the inverted repeats at processed ends (but not full-length coding ends), yet statistical analysis shows that it is virtually impossible for the inverted repeats to be polymerized by TdT. Third, TdT additions are not random. It has long been noted that TdT has a G utilization preference. In addition to the G preference, we find that TdT adds strings of purines or strings of pyrimidines at a highly significant frequency. This tendency suggests that nucleotide-stacking interactions affect TdT polymerization. All three of these features place constraints on the extent of junctional diversity in human V(D)J recombination.     

Molecular indexing of human genomic DNA

Molecular indexing sorts DNA fragments into subsets for inter-sample comparisons. Type IIS or interrupted palindrome restriction endonucleases, which result in single-stranded ends not including the original recognition sequence of the enzyme, are used to produce the fragments. The ends can then be any sequence but will always be specific for a given fragment. Fragments with particular ends are selected by ligation to a corresponding indexing adapter. We describe iterative indexing, a new process that after an initial round of indexing uses a Type IIS restriction endonuclease to expose additional sequence for further indexing. New plasmids, pINDnn, were produced for novel use as indexing adapters. Together, the plasmids index all 16 possible dinucleotides. Their large size can be increased by dimerisation in vitro and allows the isolation of indexed material by size separation. Fragments produced from human genomic DNA by Type II restriction endonucleases were sorted using six bases in total to a possible enrichment of 1920-fold. By comparison with the public human sequence databases, fidelity of indexing was shown to be high and was tolerant of repetitive sequences. Genome-wide comparisons on a candidate or non-candidate basis are made possible by this approach.     

Fusion of reverse-oriented Ds termini following abortive transposition in Arabidopsis: implications for the mechanism of Ac/Ds transposition

We studied the products of alternative transposition reactions that utilize reverse-oriented Ds termini as substrates. In this configuration, Ds transposition can generate genome rearrangements including deletions, inversions, and reciprocal translocations. In approximately half of the transposition products recovered in Arabidopsis, the termini of the reversed ends Ds element were ligated together. The sequences at these fused-end junctions suggest that the excised transposon termini form covalently closed hairpin structures. These results shed new light on the mechanism of Ac/Ds transposition.     

'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.     

A novel cDNA/PCR strategy for efficient cloning of small amounts of undefined RNA

In this report we present a strategy for generating a representative cDNA library from prohibitively low amounts of mRNA template. A defined DNA adapter, which carries an EcoRI site, is ligated to both ends of the products of a cDNA synthesis reaction. This allows low levels of cDNA to be amplified by a polymerase chain reaction. In studies with pg amounts of rabbit globin mRNA, the amplified cDNA product is shown to be full-length. Globin cDNA recombinants are positively identified in lambda gt10. The protocol should be widely applicable to mRNAs of low abundance, whose sequences have not been determined, and to limited samples from patients or animals. It may also be useful for generating representative libraries of low titer or variant viral sequences.     

Mapping of sequenced genes (700 kbp) in the restriction map of the Escherichia coli chromosome

This paper describes software (written in Pascal and running on Macintosh computers) allowing localization of unknown DNA fragments from the Escherichia coli chromosome on the restriction map established by Kohara et al. (1987). The program identifies the segment's map position using a restriction pattern analysis obtained with all, or some, of the eight enzymes used by Kohara et al. (1987). Therefore, the sequenced genes available in the EMBL library may be localized on the E. coli chromosome restriction map. This allowed correction of the map (mainly by introducing missing sites in the published maps) at the corresponding positions. Analysis of the data indicates that there is only a very low level of polymorphism, at the nucleotide level, between the E. coli K12 strains used by the various laboratories involved in DNA sequencing. The program is versatile enough to be used with other genomes.