"DIROM"--an interactive system for planning experiments on directed mutagenesis and design of artificial genes]

We present a computer system "DIROM" for oligonucleotide-directed mutagenesis and artificial gene design experiments planning and support. "DIROM" allows to search for optimal oligonucleotides according to such parameters as sufficient energy of oligonucleotide-target hybridization, secondary structure of oligonucleotide and target DNA, presence of alternative attachment sites in target DNA, terminal G/C pairs presence. Both single-stranded and double-stranded vector mutagenesis methods are implemented. It can be also used for optimal primer selection for polymerase chain reaction, sequencing etc. "DIROM" can search for both existent and potential carry out vector+target sequence construction. Both amino acid and nucleotide sequences can be operated.     

A gene-specific DNA sequencing chip for exploring molecular evolutionary change

Sequencing by hybridization (SBH) approaches to DNA sequencing face two conflicting constraints. First, in order to ensure that the target DNA binds reliably, the oligonucleotide probes that are attached to the chip array must be >15 bp in length. Secondly, the total number of possible 15 bp oligonucleotides is too large (>4¹⁵) to fit on a chip with current technology. To circumvent the conflict between these two opposing constraints, we present a novel gene-specific DNA chip design. Our design is based on the idea that not all conceivable oligonucleotides need to be placed on a chip— only those that capture sequence combinations occurring in nature. Our approach uses a training set of aligned sequences that code for the gene in question. We compute the minimum number of oligonucleotides (generally 15–30 bp in length) that need to be placed on a DNA chip to capture the variation implied by the training set using a graph search algorithm. We tested the approach in silico using cytochrome-b sequences. Results indicate that on average, 98% of the sequence of an unknown target can be determined using the approach.     

DIROM: an experimental design interactive system for directed mutagenesis and nucleic acids engineering

A computer system DIROM for oligonucleotide-directed mutagenesisand artificial gene design has been designed for better experimentalplanning and control. DIROM permits searching for optimal oligonucleotideswith respect to certain important parameters, namely sufficientenergy of oligonucleotide-target hybridization, the secondarystructure of oligonuc-tide and target DNA, the presence of alternatebinding sites in the target DNA and terminal G/C pairs. It canalso be used to plan polymerase chain reaction experiments,for optimal primer selection, in sequencing, etc. DIROM enablesone to search for both existing and potential restriction sites,to perform vector + target sequence construction. The systemconsists of a set of original algorithms that formalize theempirical knowledge of oligonucleotide action as primers.     

Hybridization methods for DNA sequencing.

I have conducted a general analysis of the practicability of using oligonucleotide hybridization to sequence DNA. Any DNA sequence may be sequenced by hybridization with a complete panel of oligonucleotides. However, sequencing DNA segments over 2 kb long requires an unrealistic number of hybridization reactions. The optimal protocol is to hybridize 7-mer or 8-mer mixed oligonucleotide probes to immobilized DNA fragments 80 bp long: should this prove impractical, hybridization of labeled 270-bp fragments to immobilized mixed 10-mers is a potential alternative. Both protocols require no more experiments to sequence large regions of DNA than conventional m13-based sequencing and are much easier to automate, thus reducing the requirements for skilled personnel. In the ideal case, hybridization sequencing reduces the number of experiments required to sequence megabase DNA by 90%.     

A computer program for choosing optimal oligonucleotides for filter hybridization, sequencing and in vitro amplification of DNA.

A method is presented for choosing optimal oligodeoxyribonucleotides as probes for filter hybridization, primers for sequencing, or primers for DNA amplification. Three main factors that determine the quality of a probe are considered: stability of the duplex formed between the probe and target nucleic acid, specificity of the probe for the intended target sequence, and self-complementarity. DNA duplex stability calculations are based on the nearest-neighbor thermodynamic values determined by Breslauer et al. [Proc. Natl. Acad. Sci. U.S.A. (1986), 83: 3746]. Temperatures of duplex dissociation predicted by the method described here were within 0.4 degrees C of the values obtained experimentally for ten oligonucleotides. Calculations for specificity of the probe and its self-complementarity are based on a simple dynamic algorithm.     

Rapid identification of clones using the same degenerate oligonucleotide mixture for both screening and sequencing

A simple and rapid strategy for distinguishing between positively hybridizing colonies and false positive-hybridization signals is described. The isolation of a specific DNA sequence depends on the ability to distinguish between a clone that contains the correct sequence and a false hybridization-positive or background signal. This procedure utilizes the same oligonucleotide mixture both as a screening probe and as a sequencing primer. The mixture of oligonucleotides is used as a primer to obtain sequence information directly from double-stranded DNA. Conditions for sequencing with oligonucleotides having up to 64-fold degeneracy are described. Since the sequence information obtained is directly adjacent to the site of oligonucleotide:DNA hybridization, it is necessary to know only a minimal length of DNA or peptide sequence to both design oligonucleotide probes and confirm the authenticity of the hybridization positives. The advantages of the degenerate oligonucleotide sequencing method include the rapid, reliable identification of authentic versus false hybridization positives made directly without subcloning into single-stranded M13 phage, without sequencing large regions of DNA, or without synthesizing sequence-specific primers.     

Novel approach for the effective determination of DNA scission site using the Sanger method

This paper describes the effective determination of DNA scission site using a novel approach that is based on the Sanger method of nucleotide sequencing. The DNA scission site is determined by contrast with the nucleotide sequence of the DNA. Here, instead of the traditional Maxam-Gilbert method for the determination of the DNA sequence, we utilized the Sanger method and studied its effectiveness in the determination of DNA scission sites. Using this method, the determination of DNA scission site becomes more facile and exact. And the total time for the determination is reduced nearly by half in comparison to the Maxam-Gilbert method. Further advantages of this novel approach include the reduced risks of radiation exposure for researchers and contamination of the apparatus.     

Microarrays for identifying binding sites and probing structure of RNAs

Oligonucleotide microarrays are widely used in various biological studies. In this review, application of oligonucleotide microarrays for identifying binding sites and probing structure of RNAs is described. Deep sequencing allows fast determination of DNA and RNA sequence. High-throughput methods for determination of secondary structures of RNAs have also been developed. Those methods, however, do not reveal binding sites for oligonucleotides. In contrast, microarrays directly determine binding sites while also providing structural insights. Microarray mapping can be used over a wide range of experimental conditions, including temperature, pH, various cations at different concentrations and the presence of other molecules. Moreover, it is possible to make universal microarrays suitable for investigations of many different RNAs, and readout of results is rapid. Thus, microarrays are used to provide insight into oligonucleotide sequences potentially able to interfere with biological function. Better understanding of structure–function relationships of RNA can be facilitated by using microarrays to find RNA regions capable to bind oligonucleotides. That information is extremely important to design optimal sequences for antisense oligonucleotides and siRNA because both bind to single-stranded regions of target RNAs.     

High-fidelity target sequencing of individual molecules identified using barcode sequences: de novo detection and absolute quantitation of mutations in plasma cell-free DNA from cancer patients

Circulating tumour DNA (ctDNA) is an emerging field of cancer research. However, current ctDNA analysis is usually restricted to one or a few mutation sites due to technical limitations. In the case of massively parallel DNA sequencers, the number of false positives caused by a high read error rate is a major problem. In addition, the final sequence reads do not represent the original DNA population due to the global amplification step during the template preparation. We established a high-fidelity target sequencing system of individual molecules identified in plasma cell-free DNA using barcode sequences; this system consists of the following two steps. (i) A novel target sequencing method that adds barcode sequences by adaptor ligation. This method uses linear amplification to eliminate the errors introduced during the early cycles of polymerase chain reaction. (ii) The monitoring and removal of erroneous barcode tags. This process involves the identification of individual molecules that have been sequenced and for which the number of mutations have been absolute quantitated. Using plasma cell-free DNA from patients with gastric or lung cancer, we demonstrated that the system achieved near complete elimination of false positives and enabled de novo detection and absolute quantitation of mutations in plasma cell-free DNA.     

A relative-entropy algorithm for genomic fingerprinting captures host-phage similarities

The degeneracy of codons allows a multitude of possible sequences to code for the same protein. Hidden within the particular choice of sequence for each organism are over 100 previously undiscovered biologically significant, short oligonucleotides (length, 2 to 7 nucleotides). We present an information-theoretic algorithm that finds these novel signals. Applying this algorithm to the 209 sequenced bacterial genomes in the NCBI database, we determine a set of oligonucleotides for each bacterium which uniquely characterizes the organism. Some of these signals have known biological functions, like restriction enzyme binding sites, but most are new. An accompanying scoring algorithm is introduced that accurately (92%) places sequences of 100 kb with their correct species among the choice of hundreds. This algorithm also does far better than previous methods at relating phage genomes to their bacterial hosts, suggesting that the lists of oligonucleotides are "genomic fingerprints" that encode information about the effects of the cellular environment on DNA sequence. Our approach provides a novel basis for phylogeny and is potentially ideally suited for classifying the short DNA fragments obtained by environmental shotgun sequencing. The methods developed here can be readily extended to other problems in bioinformatics.     

A new approach for the detection of DNA sequences in amplified nucleic acids by a surface plasmon resonance biosensor

In this paper, a simple and useful approach for DNA sensing based on surface plasmon resonance (SPR) transduction is reported. A new DNA sample pre-treatment has been optimised to allow fast and simple detection of hybridisation reaction between a target sequence in solution and a probe immobilised on the sensing surface. This pre-treatment consisted in a denaturation procedure of double stranded DNA containing the target sequence and was based on an high temperature treatment (95 degrees C, 5 min) followed by a 1 min incubation with small oligonucleotides. The oligonucleotides are designed to prevent the re-hybridising of the denatured strands, while enabling the target sequence to bind the immobilised probe. The important parameters of the procedure, i.e. incubation time, length and concentration of the oligonucleotides, have been studied in detail. The optimised DNA denaturation procedure has been successfully applied to the detection of amplified DNA with a commercially available SPR biosensor (Biacore X). DNA samples extracted from plant and human blood were tested after amplification by polymerase chain reaction (PCR).     

Determination of optimal sites of antisense oligonucleotide cleavage within TNFalpha mRNA

Antisense oligonucleotides provide a powerful tool in order to determine the consequences of the reduced expression of a selected target gene and may include target validation and therapeutic applications. Methods of predicting optimum antisense sites are not always effective. We have compared the efficacy of antisense oligonucleotides, which were selected in vitro using random combinatorial oligonucleotide libraries of differing length and complexity, upon putative target sites within TNFα mRNA. The relationship of specific target site accessibility and oligonucleotide efficacy with respect to these parameters proved to be complex. Modification of the length of the recognition sequence of the oligonucleotide library illustrated that independent target sites demonstrated a preference for antisense oligonucleotides of a defined and independent optimal length. The efficacy of antisense oligonucleotide sequences selected in vitro paralleled that observed in phorbol 12-myristate 13-acetate (PMA)-activated U937 cells. The application of methylphosphonate:phosphodiester chimaeric oligonucleotides to U937 cells reduced mRNA levels to up to 19.8% that of the untreated cell population. This approach provides a predictive means to profile any mRNA of known sequence with respect to the identification and optimisation of sites accessible to antisense oligonucleotide activity.     

Insertion site specificity of the transposon Tn3.

The Tn3-deletion method [Davies and Hutchison, Nucleic Acids Res. 19, 5731-5738, (1991)] was used to sequence a 9.4 kb DNA fragment. Transpositional 'warm' spots were not a limiting factor but a 935 bp 'cold' spot was completed using a synthetic oligonucleotide primer. Two hundred and twenty three miniTn3 insertion sites from three sequencing projects were aligned and a 19 bp asymmetric consensus site was identified. There is no absolute sequence requirement at any position in this consensus, so insertion occurs promiscuously (approximately 37% of sites are potential targets). In our sequencing projects, multiply targeted sites always closely matched the consensus, although not all close matches were targeted frequently. The 935 bp cold spot showed no unusual features when analysed with the consensus sequence. The consensus can be used to accurately predict likely insertion sites in a new sequence. Synthetic oligonucleotides based on the consensus and a known hot spot for Tn3 were mutagenised. These sequences were not hot spots in our vectors, suggesting that the primary sequence alone is not sufficient to create an insertional hot spot. We conclude that some other factor, such as DNA secondary structure, also plays an important role in target site selection for the transposon Tn3.     

Sequencing of megabase plus DNA by hybridization: theory of the method

A mismatch-free hybridization of oligonucleotides containing from 11 to 20 monomers to unknown DNA represents, in essence, a sequencing of a complementary target. Realizing this, we have used probability calculations and, in part, computer simulations to estimate the types and numbers of oligonucleotides that would have to be synthesized in order to sequence a megabase plus segment of DNA. We estimate that 95,000 specific mixes of 11-mers, mainly of the 5'(A,T,C,G)(A,T,C,G)N8(A,T,C,G)3' type, hybridized consecutively to dot blots of cloned genomic DNA fragments would provide primary data for the sequence assembly. An optimal mixture of representative libraries in M13 vector, having inserts of (i) 7 kb, (ii) 0.5 kb genomic fragments randomly ligated in up to 10-kb inserts, and (iii) tandem "jumping" fragments 100 kb apart in the genome, will be needed. To sequence each million base pairs of DNA, one would need hybridization data from about 2100 separate hybridization sample dots. Inevitable gaps and uncertainties in alignment of sequenced fragments arising from nonrandom or repetitive sequence organization of complex genomes and difficulties in cloning "poisonous" sequences in Escherichia coli, inherent to large sequencing by any method, have been considered and minimized by choice of libraries and number of subclones used for hybridization. Because it is based on simpler biochemical procedures, our method is inherently easier to automate than existing sequencing methods. The sequence can be derived from simple primary data only by extensive computing. Phased experimental tests and computer simulations increasing in complexity are needed before accurate estimates can be made in terms of cost and speed of sequencing by the new approach. Nevertheless, sequencing by hybridization should show advantages over existing methods because of the inherent redundancy and parallelism in its data gathering.     

Optimizing the design of oligonucleotides for homology directed gene targeting

Background

Gene targeting depends on the ability of cells to use homologous recombination to integrate exogenous DNA into their own genome. A robust mechanistic model of homologous recombination is necessary to fully exploit gene targeting for therapeutic benefit.

Methodology/Principal Findings

In this work, our recently developed numerical simulation model for homology search is employed to develop rules for the design of oligonucleotides used in gene targeting. A Metropolis Monte-Carlo algorithm is used to predict the pairing dynamics of an oligonucleotide with the target double-stranded DNA. The model calculates the base-alignment between a long, target double-stranded DNA and a probe nucleoprotein filament comprised of homologous recombination proteins (Rad51 or RecA) polymerized on a single strand DNA. In this study, we considered different sizes of oligonucleotides containing 1 or 3 base heterologies with the target; different positions on the probe were tested to investigate the effect of the mismatch position on the pairing dynamics and stability. We show that the optimal design is a compromise between the mean time to reach a perfect alignment between the two molecules and the stability of the complex.

Conclusion and Significance

A single heterology can be placed anywhere without significantly affecting the stability of the triplex. In the case of three consecutive heterologies, our modeling recommends using long oligonucleotides (at least 35 bases) in which the heterologous sequences are positioned at an intermediate position. Oligonucleotides should not contain more than 10% consecutive heterologies to guarantee a stable pairing with the target dsDNA. Theoretical modeling cannot replace experiments, but we believe that our model can considerably accelerate optimization of oligonucleotides for gene therapy by predicting their pairing dynamics with the target dsDNA.     

Site-specific mutagenesis method which completely excludes wild-type DNA from the transformants.

A highly efficient site-specific mutagenesis method has been devised to exclude wild-type DNA from incorporation into the transformed cells. Two complementary oligonucleotides, corresponding to a target sequence of a DNA molecule and containing an insertion mutation which created an endonuclease restriction site, were synthesized. By using the wild-type DNA molecule flanked by two restriction sites on each side of the target region as a template, the two oligonucleotide primers were extended, enriched, and isolated. The extended products, in turn, were used as templates in a polymerase chain reaction to obtain a mutagenized double-stranded DNA fragment which was conveniently cloned into plasmids by using the flanking restriction sites. Escherichia coli cells transformed by these plasmids were subject to large-scale analysis. One hundred percent of the transformants examined by colony hybridization, restriction enzyme analysis, and DNA sequencing were found to contain the mutant DNA sequence.