DNA Hairpin Molecules Research Articles

Binding Kinetics of DNA Intercalation by Small Rhodium Complexes

We characterize the kinetics and DNA binding properties of the small rhodium based molecules, [Rh(bpy)2(chrysi)]3+ (chrysi) and [Rh(bpy)2(phi)]3+ (phi). Both motifs bind to DNA. Phi contains an aromatic ring system known to intercalate between adjacent bases in a fairly sequence-neutral manner. Intercalation is weaker for chrysi due to the presence of an additional aromatic ring. Instead, chrysi has been shown to bind to DNA mismatches preferentially, ejecting and replacing the bases along the helical stack. We used optical tweezers on single DNA hairpin and long DNA molecules. Hairpin unfolding experiments on mismatch-containing sequences show that chrysi binds preferentially to sequences containing mismatches, stabilizing the hairpin. Long double stranded DNA was stretched at a constant external force in the presence of the ligand and a change in DNA extension was observed to be driven directly by ligand intercalation. Under the influence of increasing force, intercalation of the phi ligand into matched DNA is observed as expected. Surprisingly this intercalation into matched DNA was also observed for the chrysi ligand. Further analysis revealed the dissociation constants (Kd) of the chrysi and phi complexes to be on the order of 1 μM, though phi showed a significantly stronger intercalating affinity than chrysi.

Transition-Path Probability as a Test of Reaction-Coordinate Quality Reveals DNA Hairpin Folding Is a One-Dimensional Diffusive Process.

Chemical reactions are typically described in terms of progress along a reaction coordinate. However, the quality of reaction coordinates for describing reaction dynamics is seldom tested experimentally. We applied a framework for gauging reaction-coordinate quality based on transition-path analysis to experimental data for the first time, looking at folding trajectories of single DNA hairpin molecules measured under tension applied by optical tweezers. The conditional probability for being on a reactive transition path was compared with the probability expected for ideal diffusion over a 1D energy landscape based on the committor function. Analyzing measurements and simulations of hairpin folding where end-to-end extension is the reaction coordinate, after accounting for instrumental effects on the analysis, we found good agreement between transition-path and committor analyses for model two-state hairpins, demonstrating that folding is well-described by 1D diffusion. This work establishes transition-path analysis as a powerful new tool for testing experimental reaction-coordinate quality.

Dual-Beam Fluorescence Correlation Spectroscopy of DNA Hairpin Folding-Unfolding Reaction at Various Temperatures and Salt Concentrations

Fluorescence Correlation Spectroscopy has been used to study the conformational dynamics of DNA hairpin molecules of various loop lengths in aqueous solution. In a recent development, we are also able to monitor the temperature dependence of the processes of interest. In our 2-beam approach, the analyte molecules flow through an electrophoresis capillary under the influence of pressure driven flow or electrophoretic flow and are probed by two spatially offset probe volumes in such a way that the molecules flow sequentially from one probe volume to the next. A pair of dye-quencher molecules attached to DNA or RNA hairpin structures give rise to fluorescence fluctuations due to folding-unfolding dynamics of the hairpins. Fluorescence fluctuations from the probe volumes are analyzed using auto-correlation and cross-correlation analysis. In this way, we are able to resolve fluorescence fluctuation time constants arising from diffusion, flow, triplet blinking, and conformational fluctuations. Using a temperature controlling stage we can fix the temperature to within 0.2 degrees in the range 5 - 95 degrees Celsius, unaffected by the flow rate of the solution in the capillary. In this presentation, we will discuss recent results from our study of DNA hairpins of different loop lengths, at different temperatures and Sodium ion concentrations. We will also discuss binding and unbinding of counterions to individual nucleotides as they flow through the capillary under the influence of an applied electric field. Emphasis will be placed on how the desired information can be extracted using our unique approach to fluorescence correlation spectroscopy.

Single Molecule Measurements of the Unfolding Behavior of Diverse DNA Hairpin Assemblies

Kinetic and thermodynamic effects of small structural changes to the DNA duplex can be studied on a single molecule level using optical tweezers. Short DNA hairpins, which can be synthesized to contain any sequence or structure of interest, serve as our model for double stranded DNA duplexes. To facilitate pulling with optical tweezers, the hairpins are attached to long flanking DNA “handles,” which themselves are chemically attached to beads. The forces required to fully unfold the hairpin constructs are directly related to the thermodynamic stability of the hairpin structure, allowing us to quantify the effects of mismatches, lesions, intercalator binding, and other non-canonical structures that disturb the stability of double-stranded DNA. The first step in this project is to synthesize, purify, and characterize hairpin assemblies with specific sequences, utilizing a combination of solid-phase organic synthesis and molecular biology. Then, individual DNA hairpin molecules are stretched with optical tweezers. Changes in distance and force reveal that fully-matched Watson-Crick duplex hairpins unfold and refold neatly in discrete, concerted events, whereas mismatched and bubbled hairpins open and close with lower force and greater force variability. Although the overall stability of the structures is predicted well by mfold calculations, the single molecule studies allow measurement of the barrier to unfolding as well as the distance to the transition state. After validating the method for DNA alone, changes in the transition state and hairpin stability in the presence of DNA binding ligands can then be probed directly.

Unfolding kinetics of periodic DNA hairpins

DNA hairpin molecules with periodic base sequences can be expected to exhibit a regular coarse-grained free energy landscape (FEL) as a function of the number of open base pairs and applied mechanical force. Using a commonly employed model, we first analyze for which types of sequences a particularly simple landscape structure is predicted, where forward and backward energy barriers between partly unfolded states are decreasing linearly with force. Stochastic unfolding trajectories for such molecules with simple FEL are subsequently generated by kinetic Monte Carlo simulations. Introducing probabilities that can be sampled from these trajectories, it is shown how the parameters characterizing the FEL can be estimated. Already 300 trajectories, as typically generated in experiments, provide faithful results for the FEL parameters.

Yoctoliter Thermometry for Single‐Molecule Investigations: A Generic Bead‐on‐a‐Tip Temperature‐Control Module

A new temperature-jump (T-jump) strategy avoids photo-damage of individual molecules by focusing a low-intensity laser on a black microparticle at the tip of a capillary. The black particle produces an efficient photothermal effect that enables a wide selection of lasers with powers in the milliwatt range to achieve a T-jump of 65 °C within milliseconds. To measure the temperature in situ in single-molecule experiments, the temperature-dependent mechanical unfolding of a single DNA hairpin molecule was monitored by optical tweezers within a yoctoliter volume. Using this bead-on-a-tip module and the robust single-molecule thermometer, full thermodynamic landscapes for the unfolding of this DNA hairpin were retrieved. These approaches are likely to provide powerful tools for the microanalytical investigation of dynamic processes with a combination of T-jump and single-molecule techniques.

Combined versatile high-resolution optical tweezers and single-molecule fluorescence microscopy

Optical trapping and single-molecule fluorescence are two major single-molecule approaches. Their combination has begun to show greater capability to study more complex systems than either method alone, but met many fundamental and technical challenges. We built an instrument that combines base-pair resolution dual-trap optical tweezers with single-molecule fluorescence microscopy. The instrument has complementary design and functionalities compared with similar microscopes previously described. The optical tweezers can be operated in constant force mode for easy data interpretation or in variable force mode for maximum spatiotemporal resolution. The single-molecule fluorescence detection can be implemented in either wide-field or confocal imaging configuration. To demonstrate the capabilities of the new instrument, we imaged a single stretched λ DNA molecule and investigated the dynamics of a DNA hairpin molecule in the presence of fluorophore-labeled complementary oligonucleotide. We simultaneously observed changes in the fluorescence signal and pauses in fast extension hopping of the hairpin due to association and dissociation of individual oligonucleotides. The combined versatile microscopy allows for greater flexibility to study molecular machines or assemblies at a single-molecule level.

Micropatterned aptasensors for continuous monitoring of cytokine release from human leukocytes.

We report the development of a microdevice for detecting local interferon gamma (IFN-γ) release from primary human leukocytes in real time. Our microdevice makes use of miniature aptamer-modified electrodes integrated with microfluidics to monitor cellular production of IFN-γ. The aptamer species consists of a DNA hairpin molecule with thiol groups on the 3'-end for self-assembly onto Au electrodes. A redox reporter is covalently attached at the 5'-end for electrochemical sensing. This aptasensor has excellent sensitivity for IFN-γ (<60 pM detection limit) and responds to the target analyte in real time without additional washing or labeling steps. Aptamer-functionalized electrode arrays are fabricated on glass slides containing poly(ethylene glycol) (PEG) hydrogel patterns designed to expose glass regions adjacent to electrodes while protecting the remainder of the surface from nonspecific adsorption. The micropatterned substrates are integrated with PDMS microfluidic channels and incubated with T-cell-specific antibodies (Ab) (anti-CD4). Upon injection of blood, leukocytes are bound to Ab-modified glass regions in proximity to aptasensors. Cytokine release from captured cells is triggered by mitogenic activation and detected at the aptamer-modified electrodes using square wave voltammetry (SWV). The IFN-γ signal is monitored in real time with signal appearing as early as 15 min poststimulation from as few as 90 T cells. The observed IFN-γ release profiles are used to calculate an initial IFN-γ production rate of 0.0079 pg cell(-1) h(-1) upon activation. The work described here represents an important step toward development of aptasensors for immune cell analysis and blood-based diagnostics.

Magnetically Driven Single DNA Nanomotor

Magnetically driven DNA nanomotors: one end of a set of DNA hairpins is immobilized on a glass surface and the other is conjugated to magnetic particles. The DNA hairpin molecules are opened by an external magnetic field gradient, and subsequently closed through DNA hybridization after the removal of the magnetic field. The separation of the ends of the DNA hairpin during opening is interpreted as the power stroke of the nanomotor and the hybridization of the hairpin as the recovery stroke.

Sequence-dependent unfolding kinetics of DNA hairpins studied by nanopore forcespectroscopy

Nanopore force spectroscopy is used to study the unzipping kinetics of two DNA hairpinmolecules with a 12 base pair long stem containing two contiguous stretches of six GC andsix AT base pairs in interchanged order. Even though the thermodynamic stabilities ofthe two structures are nearly the same, they differ greatly in their unzippingkinetics. When the GC segment has to be broken before the AT segment, theunfolding rate is orders of magnitude smaller than in the opposite case. We alsoinvestigated hairpins with stem regions consisting only of AT or GC base pairs. Thepure AT hairpins translocate much faster than the other hairpins, whereas thepure GC hairpins translocate on similar timescales to the hairpins with only aninitial GC segment. For each hairpin, nanopore force spectroscopy is performed fordifferent loading rates and the resulting unzipping distributions are mathematicallytransformed to a master curve that yields the unfolding rate as a function of appliedvoltage. This is compared with a stochastic model of the unfolding process for thetwo sequences for different voltages. The results can be rationalized in termsof the different natures of the free energy landscapes for the unfolding process.

Nanopore Translocation Experiments in Microemulsion Droplets

We show that a novel bilayer formation technique based on microemulsion droplets introduced by Bayley and coworkers can be utilized to perform nanopore DNA translocation experiments. In this technique, a bilayer is formed between two touching emulsion droplets.By reduction of their contact area, we were able to reduce the measurementnoise sufficiently to perform single molecule translocation and even nanopore force spectroscopy experiments. We applied this technique to the translocation of DNA hairpin molecules, but also to a G-quadruplex DNA structure, which has not been characterized using nanopore force spectroscopy before. From the data, the unfolding rates of these DNA structures are extracted and compared with those obtained with other single molecule techniques.

Single-molecule Studies of RNA Unzipping Kinetics Using Nanopores

The α-Hemolysin protein pore has been previously used to study the unzipping kinetics of DNA duplexes, taking advantage of its sub-2nm pore constriction, which permits single stranded nucleic acids but not double stranded structures. Here we present the first extensive nanopore study of RNA unzipping kinetics as a function of voltage and temperature, and compare it with DNA unzipping of molecules with identical primary sequence. Our studies reveal clear differences between RNA and DNA, having similar bulk melting properties, highlighting the nanopore ability to probe subtle differences in nucleic acids' free energy and their interactions with the pore. In addition, we find that RNA hybrids with different overhang sequences display large difference in both unzipping times and current blockage signals. These results may be interpreted in the context of strong interactions between poly-A tail and the pore. Our work sheds light on the mechanics of nanopore unzipping and its sensitivity to small differences in nucleic acid base pairing stability. We currently in the process of developing a theoretical model to explain these observations, which will also set the stage for further studies involving multiple hairpin structures of RNA, and to more complicated and interesting biological problems, involving RNA-helicase interactions.1. Mathe, J., Visram, H., Viasnoff, V., Rabin, Y. & Meller, A. (2004) Nanopore unzipping of individual DNA hairpin molecules. Biophys J 87, 3205-3212.2. Dudko, O., Mathe, J., Szabo, A., Meller, A. & Hummer, G. (2007) Extracting kinetics from single-molecule force spectroscopy: Nanopore unzipping of DNA hairpins. Biophys. J. 92, 4188-4195.

Simultaneous Alternating and Direct Current Readout of Protein Ion Channel Blocking Events Using Glass Nanopore Membranes

Alternating current (ac) phase-sensitive detection is used to measure the conductance of the ion channel alpha-hemolysin (alphaHL), while simultaneously applying a direct current (dc) bias to electrostatically control the binding affinity and kinetics of charged molecules within the protein lumen. Ion channel conductance was recorded while applying a 10-20 mV rms, 1-2 kHz bias across a single alphaHL protein inserted in a 1,2-diphytanoyl-sn-glycero-3-phosphocholine lipid bilayer that is suspended across the orifice (100-500 nm radius) of a glass nanopore membrane. Step changes in the ac ion channel conductance with a temporal response (t(10-90)) of 1.5 ms and noise amplitude of approximately 2 pA were obtained using a low-noise potentiostat and a lock-in amplifier. These conditions were used to monitor the reversible and stochastic binding of heptakis-(6-O-sulfo)-beta-cyclodextrin and a nine base pair DNA hairpin molecule to the ion channel. Alternating current methodology allows the binding kinetics and affinity between the protein ion channel and analyte to be investigated as a function of the dc bias, including ion channel conductance measurements in the absence of a dc bias.

Probing the Complete Folding Trajectory of a DNA Hairpin Using Dual Beam Fluorescence Fluctuation Spectroscopy

The conformational fluctuations of dye-quencher labeled DNA hairpin molecules in aqueous solution were investigated using dual probe beam fluorescence fluctuation spectroscopy. The measurements revealed the flow and diffusion times of the DNA molecules through two spatially offset optical probe regions, the absolute and relative concentrations of each conformational substate of the DNA, and the kinetics of the DNA hairpin folding and unfolding reactions in the 1 micros to 10 ms time range. A DNA hairpin containing a 21-nucleotide polythymine loop and a 4-base pair stem exhibited double exponential relaxation kinetics, with time constants of 84 and 393 micros. This confirms that folding and melting of the DNA hairpin structure is not a two state process but proceeds by way of metastable intermediate states. The fast time constant corresponds to formation and unfolding of an intermediate, and the slow time constant is due to formation and disruption of the fully base-paired stem. This is consistent with a previous study of a similar DNA hairpin with a 5-base pair stem, in which the fast reaction was attributed to the fluctuations of an intermediate DNA conformation [J. Am. Chem. Soc. 2006, 128, 1240-1249]. In that case, reactions involving the native conformation could not be observed directly due to the limited observation time range of the fluorescence correlation spectroscopy experiment. The intermediate states of the DNA hairpins are suggested to be due to a collapsed ensemble of folded hairpins containing various partially folded or misfolded conformations.

A novel, fast, HMM-with-Duration implementation – for application with a new, pattern recognition informed, nanopore detector

BackgroundHidden Markov Models (HMMs) provide an excellent means for structure identification and feature extraction on stochastic sequential data. An HMM-with-Duration (HMMwD) is an HMM that can also exactly model the hidden-label length (recurrence) distributions – while the regular HMM will impose a best-fit geometric distribution in its modeling/representation.ResultsA Novel, Fast, HMM-with-Duration (HMMwD) Implementation is presented, and experimental results are shown that demonstrate its performance on two-state synthetic data designed to model Nanopore Detector Data. The HMMwD experimental results are compared to (i) the ideal model and to (ii) the conventional HMM. Its accuracy is clearly an improvement over the standard HMM, and matches that of the ideal solution in many cases where the standard HMM does not. Computationally, the new HMMwD has all the speed advantages of the conventional (simpler) HMM implementation. In preliminary work shown here, HMM feature extraction is then used to establish the first pattern recognition-informed (PRI) sampling control of a Nanopore Detector Device (on a "live" data-stream).ConclusionThe improved accuracy of the new HMMwD implementation, at the same order of computational cost as the standard HMM, is an important augmentation for applications in gene structure identification and channel current analysis, especially PRI sampling control, for example, where speed is essential. The PRI experiment was designed to inherit the high accuracy of the well characterized and distinctive blockades of the DNA hairpin molecules used as controls (or blockade "test-probes"). For this test set, the accuracy inherited is 99.9%.

Sequence-dependent gating of an ion channel by DNA hairpin molecules.

DNA hairpins produce ionic current signatures when captured by the alpha-hemolysin nano-scale pore under conditions of single molecule electrophoresis. Gating patterns produced by individual DNA hairpins when captured can be used to distinguish differences of a single base pair or even a single nucleotide [Vercoutere,W.A. et al. (2003) Nucleic Acids Res., 31, 1311–1318]. Here we investigate the mechanism(s) that may account for the ionic current gating signatures. The ionic current resistance profile of conductance states produced by DNA hairpin molecules with 3–12 bp stems showed a plateau in resistance between 10 and 12 bp, suggesting that hairpins with 10–12 bp stems span the pore vestibule. DNA hairpins with 9–12 bp stems produced gating signatures with the same relative conductance states. Systematic comparison of the conductance state dwell times and apparent activation energies for a series of 9–10 bp DNA hairpins suggest that the 3′ and 5′ ends interact at or near the limiting aperture within the vestibule of the alpha-hemolysin pore. The model presented may be useful in predicting and interpreting DNA detection using nanopore detectors. In addition, this well-defined molecular system may prove useful for investigating models of ligand-gated channels in biological membranes.

Rapid Fabrication of Uniformly Sized Nanopores and Nanopore Arrays for Parallel DNA Analysis

Nanometer-sized pores can be used to detect and characterize biopolymers, such as DNA, RNA, and polypeptides, with single-molecule resolution. Experiments performed with the 1.5 nm pore a-hemolysin (a-HL) demonstrated that singlestranded DNA and RNA molecules can be electrophoretically threaded through a pore, and that the ion current flowing through the pore contains information about the biopolymer sequence: its type, length, and secondary structure. The a-HL nanopore has been used to study the unzipping kinetics of DNA hairpin molecules under stationary or time-varying forces, to detect DNA hybridization kinetics, and to study the interaction of DNA with bound proteins using nanopore force spectroscopy. In addition, a-HL can be biochemically modified for various sensing tasks, such as analyte detection and ligand–receptor interactions. Solid-state nanopores can be fabricated in thin Si3N4 and SiO2 membranes, using either an Ar beam or an electron beam (e-beam) in a transmission electron microscope (TEM), as well as in a variety of materials using other techniques. Solid-state nanopores offer several advantages over phospholipid-embedded protein channels, namely, their size can be tuned with nanometer precision and they exhibit an increased mechanical, chemical, and electrical stability. Recent studies using solid-state pores have begun to emerge, demonstrating the detection of single-stranded and double-stranded DNA molecules. A major advantage of solid-state nanopores is that they can, in principle, be integrated into devices compatible with other detection schemes in addition to ion current measurements. In particular, optical-based methods offer straightforward parallelism through the simultaneous probing of many nanopores. Optical methods for sensing single molecules can be implemented by labeling the biomolecules and/or the nanopores. Although protein pores embedded in a phospholipid bilayer can be interrogated optically to detect single molecules, a stable, long-timescale probing is very complicated since the pores readily diffuse in the bilayer, leading to aggregation and destabilization of the membrane. In contrast, nanopores fabricated in solid-state materials are static, and are therefore more compatible with optical probing. In this paper, we extend state-of-the-art techniques by demonstrating the rapid fabrication of finely tuned nanopores and nanopore arrays. The nanopores were fabricated in thin Si3N4 films using the intense e-beam of a field-emission TEM. By maximizing the e-beam density at the specimen we achieved a nearly fivefold decrease in the fabrication time of a single nanopore (ca. 30 s). Investigation of pore contraction/expansion dynamics under different irradiation conditions enabled nanopore fabrication in the range of 2–20 nm with exceptional size control (<0.5 nm variability). Since the nanopores were fabricated sequentially (i.e., using one e-beam), both the reduction in fabrication time and size control were crucial for the manufacturing of nanopore arrays. The 3D nanopore shape was extensively characterized by performing TEM tomography, as well as by ion-current measurements through the pores. Finally, the detection of double-stranded DNA molecules through 4 nm diameter nanopores was demonstrated by monitoring their translocation under an applied bias. The starting materials for TEM processing were either fabricated in house or by Protochips Inc. (Raleigh, NC), using the following procedure: low-pressure chemical vapor deposition (LPCVD) was used to form a Si3N4 film (20 or 50 nm thick) on one side of a 500 lm thick Si wafer. A 100 lm× 100 lm window was then fabricated in the wafer using photolithography and standard wet-etching. Nanopore fabrication was then carried out in the thin Si3N4 membrane using a JEOL 2010F field-emission TEM. Alignment of the e-beam involved the adjustment of the condenser stigmatism to the familiar triangle-shaped beam, using a large condenser aperture. The resulting electron-energy-density distribution displayed a threefold aberration. The condenser lens was then used to fully converge the beam to an intense point with a triangular halo of low intensity. The column was then aligned using standard high-resolution transmission electron microscopy alignment procedures. After the alignment procedure, nanopores with diameters (d) in a range from 3 to 6 nm were directly drilled using an e-beam intensity of ca. 2.5× 10 e nm and a CO M M U N CA IO N

A Three-State Mechanism for DNA Hairpin Folding Characterized by Multiparameter Fluorescence Fluctuation Spectroscopy

The folding of a dye-quencher labeled DNA hairpin molecule was investigated using fluorescence autocorrelation and cross-correlation spectroscopy (FCS) and photon counting histogram analysis (PCH). The autocorrelation and cross-correlation measurements revealed the flow and diffusion times of the DNA molecules through two spatially offset detection volumes, the relaxation time of the folding reaction, and the total concentration of DNA molecules participating in the reaction. The PCH measurements revealed the equilibrium distribution of DNA molecules in folded and unfolded conformations and the specific brightnesses of the fluorophore in each conformational state. These measurements were carried out over a range of NaCl concentrations, from those that favored the open form of the DNA hairpin to those that favored the closed form. DNA melting curves obtained from each sample were also analyzed for comparison. It was found that the reactant concentrations were depleted as the reaction progressed and that the equilibrium distributions measured by FCS and PCH deviated from those obtained from the melting curve analyses. These observations suggest a three-state mechanism for the DNA hairpin folding reaction that involves a stable intermediate form of the DNA hairpin. The reaction being probed by FCS and PCH is suggested to be a rapid equilibrium between open and intermediate conformations. Formation of the fully closed DNA hairpin is suggested to occur on a much longer time scale than the FCS and PCH measurement time. The closed form of the hairpin thus serves as a sink into which the reactants are depleted as the reaction progresses.

Folding and Unfolding Kinetics of DNA Hairpins in Flowing Solution by Multiparameter Fluorescence Correlation Spectroscopy

Dynamic equilibrium between the folded and unfolded conformations of single stranded DNA hairpin molecules containing polythymine hairpin loops was investigated using simultaneous two-beam fluorescence cross-correlation spectroscopy and single beam autocorrelation spectroscopy. The hairpins were end-labeled with a fluorescent dye and a quencher, such that folding and unfolding of the DNA hairpin primary structure caused the dye fluorescence to fluctuate on the same characteristic time scale as the folding and unfolding reaction. These fluctuations were observed as the molecules flowed sequentially between two spatially offset, microscopic detection volumes. Cross-correlation analysis of fluorescence from the two detection volumes revealed the translational diffusion and flow properties of the hairpins, as well as the average molecular occupancy of the two volumes. Autocorrelation analysis of the fluorescence from the individual detection volumes revealed the kinetics of hairpin folding and unfolding, with the parameters relating to diffusion, flow, and molecular occupancy constrained to the values determined from the cross-correlation analysis. This allowed unambiguous characterization of the folding and unfolding kinetics, without the need to determine the hydrodynamic properties by analyzing a separate control sample. The analysis revealed nonexponential relaxation kinetics and DNA size-dependent folding times characteristic of dynamic heterogeneity in the DNA hairpin-forming mechanism.

Hybridization chain reaction

A new technique, based on a chain reaction of recognition and hybridization events between two sets of DNA hairpin molecules, offers an enzyme-free alternative for the rapid detection of specific DNA sequences.