Search

We report the use of an array of electrically gated ~200 nm solid-state pores as nanofluidic transistors to manipulate the capture and passage of DNA. The devices are capable of reversibly altering the rate of DNA capture by over 3 orders of magnitude using sub-1 V biasing of a gate electrode. This efficient gating originates from the counter-balance of electrophoresis and electroosmosis, as revealed by quantitative numerical simulations. Such a reversible electronically tunable biomolecular switch may be used to manipulate nucleic acid delivery in a fluidic circuit, and its development is an important first step toward active control of DNA motion through solid-state nanopores for sensing applications.

We combined components of a previous assay referred to as Molecular Inversion Probe (MIP) with a complete gap filling strategy, creating a versatile powerful one-primer multiplex amplification system. As a proof-of-concept, this novel method, which employs a Connector Inversion Probe (CIPer), was tested as a genetic tool for pathogen diagnosis, typing, and antibiotic resistance screening with two distinct systems: i) a conserved sequence primer system for genotyping Human Papillomavirus (HPV), a cancer-associated viral agent and ii) screening for antibiotic resistance mutations in the bacterial pathogen Neisseria gonorrhoeae. We also discuss future applications and advances of the CIPer technology such as integration with digital amplification and next-generation sequencing methods. Furthermore, we introduce the concept of two-dimension informational barcodes, i.e. "multiplex multiplexing padlocks" (MMPs). For the readers' convenience, we also provide an on-line tutorial with user-interface software application CIP creator 1.0.1, for custom probe generation from virtually any new or established primer-pairs.

The analysis of short tandem repeats (STRs) plays an important role in forensic science, human identification, genetic mapping, and disease diagnostics. Traditional STR analysis utilizes gel- or column-based approaches to analyze DNA repeats. Individual STR alleles are separated and distinguished according to fragment length; thus the assay is generally hampered by its low multiplex capacity. However, use of DNA microarray would employ a simple hybridization and detection for field forensics and biology. Here we demonstrate a rapid, highly sensitive method for STR analysis that utilizes DNA microarray technology. We describe two adaptations to accomplish this: the use of competitive hybridization to remove unpaired ssDNA from an array and the use of neural network classification to automate the analysis. The competitive displacement technique mimics the branch migration process that occurs during DNA recombination. Our technique will facilitate the rapid deduction of identity, length, and number of repeats for the multiple STRs in an unknown DNA sample.

Rapid, sequence-specific DNA detection is essential for applications in medical diagnostics and genetic screening. Electrical biosensors that use immobilized nucleic acids are especially promising in these applications because of their potential for miniaturization and automation. Current DNA detection methods based on sequencing by synthesis rely on optical readouts; however, a direct electrical detection method for this technique is not available. We report here an approach for direct electrical detection of enzymatically catalyzed DNA synthesis by induced surface charge perturbation. We discovered that incorporation of a complementary deoxynucleotide (dNTP) into a self-primed single-stranded DNA attached to the surface of a gold electrode evokes an electrode surface charge perturbation. This event can be detected as a transient current by a voltage-clamp amplifier. Based on current understanding of polarizable interfaces, we propose that the electrode detects proton removal from the 3'-hydroxyl group of the DNA molecule during phosphodiester bond formation.

We describe a novel method for rapidly identifying and distinguishing between different DNA sequences using short tandem repeat (STR) analysis and DNA microarrays. The method can be used to deduce identity, length, and number of STRs of the target molecule. We refer to this technique as the "variable-length probe array" method for STR profiling (VLPA). The method involves hybridization of the unknown STR target sequence to a DNA microarray displaying complementary probes that vary in length to cover the range of possible STRs. A post-hybridization enzymatic digestion of the DNA hybrids is then used to selectively remove labeled single-stranded regions of DNA from the microarray surface. The number of repeats in the unknown target is then deduced based on the pattern of target DNA that remains hybridized to the array. This DNA profiling technique is useful for performing forensic analysis to uniquely identify individual humans or other species.

Single DNA molecules labeled with nanoparticles can be detected by blockades of ionic current as they are translocated through a nanopipette tip formed by a pulled glass capillary. The nanopipette detection technique can provide not only tools for detection and identification of single DNA and protein molecules but also deeper insight and understanding of stochastic interactions of various biomolecules with their environment.

Pyrosequencing is a DNA sequencing technique based on sequencing-by-synthesis enabling rapid real-time sequence determination. This technique employs four enzymatic reactions in a single tube to monitor DNA synthesis. Nucleotides are added iteratively to the reaction and in case of incorporation, pyrophosphate (PPi) is released. PPi triggers a series of reactions resulting in production of light, which is proportional to the amount of DNA and number of incorporated nucleotides. Generated light is detected and recorded by a detector system in the form of a peak signal, which reflects the activity of all four enzymes in the reaction. We have developed simulations to model the kinetics of the enzymes. These simulations provide a full model for the Pyrosequencing four-enzyme system, based on which the peak height and shape can be predicted depending on the concentrations of enzymes and substrates. Simulation results are shown to be compatible with experimental data. Based on these simulations, the rate-limiting steps in the chain can be determined, and K(M) and kcat of all four enzymes in Pyrosequencing can be calculated.

Recent advances in DNA sequencing technologies, both in the form of high lane-density gels and automated capillary systems, will lead to an increased requirement for sample preparation systems that operate at low cost and high throughput. As part of the development of a fully automated sequencing system, we have developed an automated subsystem capable of producing 10,000 sequence-ready ssDNA templates per day from libraries of M13 plaques at a cost of $0.29 per sample. This Front End has been in high throughput operation since June, 1997 and has produced > 400,000 high-quality DNA templates.

Based on a high-performance liquid chromatographic pump, we have built a device that allows recirculation of DNA through a 63-microm orifice with ensuing fractionation to a minimum fragment size of approximately 300 base pairs. Residence time of the DNA fragments in the converging flow created by a sudden contraction was found to be sufficiently long to allow extension of the DNA molecules into a highly extended conformation and, hence, breakage to occur at midpoint. In most instances, 30 passages sufficed to obtain a narrow size distribution, with >90% of the fragments lying within a 2-fold size distribution. The shear rate required to achieve breakage was found to be inversely proportional to the 1.0 power of the molecular weight. Compared with a restriction digest, up to 40% of all fragments could be cloned directly, with only marginal improvements in cloning efficiency having been observed upon prior end repair with Klenow, T4 polymerase or T4 polynucleotide kinase. Sequencing revealed a fairly random distribution of the fragments.

An automated oligonucleotide synthesizer has been developed that can simultaneously and rapidly synthesize up to 96 different oligonucleotides in a 96-well microtiter format using phosphoramidite synthesis chemistry. A modified 96-well plate is positioned under reagent valve banks, and appropriate reagents are delivered into individual wells containing the growing oligonucleotide chain, which is bound to a solid support. Each well has a filter bottom that enables the removal of spent reagents while retaining the solid support matrix. A seal design is employed to control synthesis environment and the entire instrument is automated via computer control. Synthesis cycle times for 96 couplings are < 11 min, allowing a plate of 96 20-mers to be synthesized in < 5 hr. Oligonucleotide synthesis quality is comparable to commercial machines, with average coupling efficiencies routinely > 98% across the entire 96-well plate. No significant well-to-well variations in synthesis quality have been observed in > 6000 oligonucleotides synthesized to date. The reduced reagent usage and increased capacity allow the overall synthesis cost to drop by at least a factor of 10. With the development of this instrument, it is now practical and cost-effective to synthesize thousands to tens of thousands of oligonucleotides.

In reactions between linear single-stranded DNAs (ssDNAs) and circular double-stranded DNAs (dsDNAs), stable joint molecule formation promoted by the recA protein (RecA) requires negative superhelicity, a homologous end, and an RecA-ssDNA complex. Linear ssDNAs with 3'-end homology react more efficiently than linear ssDNAs with 5'-end homology. This 3'-end preference is explained by the finding that 3'-ends are more effectively coated by RecA than 5'-ends, as judged by exonuclease VII protection, and are thus more reactive. The ability of linear ssDNAs with 5'-end homology to react is improved by the presence of low concentrations of exonuclease VII. In reactions between ssDNAs and linear dsDNAs with end homology, stable joint molecule formation occurs more efficiently when the homology is at the 3'-end rather than at the 5'-end of the complementary strand. In addition, linear dsDNAs with homology at the 3'-end of the complementary strand react more efficiently with linear ssDNAs with 3'-end homology than with linear ssDNAs with 5'-end homology. The ability of linear ssDNAs with 5'-end homology to react, in the absence of single-stranded DNA-binding protein, is improved by adding 33-46 nucleotides of heterologous sequence to the 5'-end of the linear ssDNA. The poor reactivity of linear ssDNAs with 5'-end homology is explained by a lack of RecA at the 5'-ends of linear ssDNAs, which is a consequence of the polar association and dissociation of RecA.

The effects of Eco RI endonuclease-cleaved Escherichia coli and yeast (Saccharomyces cerevisiae) DNA fragments on the propagation of the lambda bacteriophage vectors containing them were determined on a nonmutanted and a PolA E. coli K12 host. Observable alterations in the growth of hybrids containing yeast DNA insertions were less frequent and less extreme than those seen in hybrids containing E. coli DNA. A lambda-E. coli hybrid was selected after extensive growth on the Pol A (deficient in polymerase I) host which also grew very well on the PolA+ host and may have resulted from some alteration in the hybrid. Hybrids selected on the PolA host gave no evidence for the expression of polymerase I activity. No lambda-yeast hybrid made from the lambdagt vector lacking lambda-specific recombination (red-) had a yield of viable bacteriophage on infection greater than two-thirds that of "wild-type" lambda.

A yeast artificial chromosome (YAC) library in Saccharomyces cerevisiae consisting of 30,000 clones with an average insert size of 0.1 megabase pair of human DNA has been generated from primary fibroblast DNA. A YAC vector was modified to enable the recovery of both ends of a human DNA insert in plasmids in Escherichia coli and to confer G418 resistance to mammalian cells. A rapid method for yeast colony hybridization was used that exploits the ability of yeast spheroplasts to regenerate in a thin layer of calcium alginate. This method permits direct replica plating and processing of colonies from the primary transformation plate to nitrocellulose filters. Yeast colony hybridization conditions have been established to identify, within a YAC library of human genomic DNA, artificial chromosomes with homology to human DNA probes of unique single-copy sequence. An artificial chromosome with a 0.1-megabase-pair insert from the human Xq28 region has been identified by hybridization to a DNA probe that detects a unique sequence near the 3' end of the factor VIII gene.

Electric fields can be manipulated by a method in which multiple electrodes are arranged along a closed contour and clamped to predetermined electric potentials. This method may be applied to a broad range of problems in the separation of macromolecules by gel electrophoresis. DNA molecules as large as 2 megabases can be well separated with a contour-clamped homogeneous electric field alternating between two orientations 120 degrees apart. The pattern of separation is independent of position in the gel, which is an advantage over previous methods. DNA less than 50 kilobases can be separated without distortion even at high voltage with a nonalternating contour-clamped homogeneous field. Decreased band broadening in DNA less than 200 bases can be achieved with a contour-clamped inhomogeneous field.

A bacteriophage lambda strain has been constructed and a method developed by which DNA from potentially any source can be covalently inserted through EcoRI cohesive ends into the middle of the lambda DNA. These hybrid DNAs can infect nonrestricting Escherichia coli cells and can then propagate as plaque-forming phage. A unique feature of this lambda strain is that extra DNA in the middle of its genome is required for plaque formation. A large number of such phages have been produced with E. coli DNA and Drosophila melanogaster DNA.

We have isolated a segment of DNA from the eukaryote Saccharomyces cerevisiae (baker's yeast) as a viable molecular hybrid of bacteriophage lambda DNA which, when integrated into the chromosome of an E. coli histidine auxotroph, allows this bacterium to grow in the absence of histidine. The nonrevertable, histidine auxotroph lacks the enzymatic activity of imidazole glycerol phosphate (IGP) dehydratase (EC 4.2.1.19). From genetic experiments, we conclude that expression of the segment of yeast DNA results in the production of a diffusible substance and that transcription necessary for the complementation is most likely initiated from the segment of eukaryotic DNA.

Dispersed repetitive DNA sequences from yeast (Saccharomyces cerevisiae) nuclear DNA have been isolated as molecular hybrids in lambdagt. Related S. cerevisiae strains show marked alterations in the size of the restriction fragments containing these repetitive DNAs. "Ty1" is one such family of repeated sequences in yeast and consists of a 5.6 kilobase (kb) sequence including a noninverted 0.25 kb sequence of another repetitious family, "delta", on each end. There are about 35 copies of Ty1 and at least 100 copies of delta (not always associated with Ty1) in the haploid genome. A few Ty1 elements are tandem and/or circular, but most are disperse and show (along with delta) some sequence divergence between repeat units. Sequence alterations involving Ty1 elements have been found during the continual propagation of a single yeast clone over the course of a month. One region with a large number of delta sequences (SUP4) also shows a high frequency of sequence alterations when different strains are compared. One of the differences between two such strains involves the presence or absence of a Ty1 element. The novel joint is at one inverted pair of delta sequences.

DNA fragments that show retarded electrophoretic mobility through polyacrylamide gels have been found in both prokaryotes and eukaryotes. In the case of kinetoplast DNA, evidence has been presented that the DNA is curved or 'bent'. Bent DNA has previously been found at the lambda and simian virus 40 (SV40) DNA replication origins. Here we show the existence of bent DNA at a yeast autonomously replicating sequence (ARS1), a putative replication origin. The bent DNA has been localized to a 40-55 base pair (bp) segment and contains six (A)3-5 stretches (that is, six poly(A) stretches, three to five nucleotides in length) phased approximately every 10.5 bp. This region contains a DNA binding site for a yeast protein factor. This site lies at the 3' end of the TRP1 gene, in a region devoid of nucleosomes, and is positioned 80 bp away from the ARS consensus sequence; removal of this region impairs ARS function in vivo. The bent DNA may be involved in transcription termination or the prevention of nucleosome assembly in this region.

A selective scheme is presented for isolating sequences capable of replicating autonomously in the yeast Saccharomyces cerevisiae. YIp5, a vector that contains the yeast gene ura3, does not transform a ura3 deletion mutant to Ura+. Hybrid YIp5-Escherichia coli DNA molecules also fail to produce transformants. However, collections of molecular hybrids between YIp5 and DNA from any of six eukaryotes tested (S. cerevisiae, Neurospora crassa, Dictyostelium discoideum Ceanhorabditis elegans, Drosophila melanogaster, and Zea mays) do transform the deletion mutant. The Ura+ transformants grow slowly, are unstable under nonselective conditions, and carry the transforming DNA as autonomously replicating, supercoiled circular molecules. Such a phenotype is qualitatively identical to that of strains transformed by molecules containing a yeast chromosomal origin of replication. Thus, these DNA hybrid molecules may contain eukaryotic origins of replication. The isolated sequences may be useful in determiing the signals controlling DNA replication in yeast and in studying both DNA replication and transformation in other eukaryotic organisms.

Excellent resolution of chromosomal DNA molecules from Saccharomyces cerevisiae, Candida albicans and Schizosaccharomyces pombe has been obtained using alternating contour-clamped homogeneous electric field (CHEF) gel electrophoresis. The largest of these molecules is greater than 5 Mb in size and is resolved after 130 hours in a 0.6% agarose gel at a field strength of 1.3 V/cm and a switching interval of 1 hour. Separation of concatamers of phage lambda DNA reveals four regions of resolution in alternating CHEF gel electrophoresis. There are two regions of good resolution in which mobility approximates a linear function of molecular weight. These are separated by a region of lower resolution and bounded at high molecular weights by a region of little or no resolution. The four regions are of practical and possibly theoretical importance.