Sequence detection system

Abstract

To provide a system and method for detecting a sequence of entity types.SOLUTION: A sequence detection system for detecting a sequence of entity types in a target including a linear sequence of entities comprises: a DNA polymerase that exhibits one or more mechanical changes when specifically interacting with the entities in the target, each entity having a type; a mechanical-change-to-signal transducer that transduces the one or more mechanical changes into an electrical signal, and includes a DNA-polymer tether attached at one end to the DNA polymerase and a reporter region of the DNA-polymer tether that lies within a constriction within a porin channel; and one or more memories of a computer including instructions stored for implementing an analysis subsystem that determines the type of each entity in the target using the electrical signal.SELECTED DRAWING: None

Description

[Technical field]

[0001] background In industrial and research environments, sequences of many different types of objects or entities are encountered. DNA sequencing and RNA sequencing are employed in many different research, diagnostic, forensic, and pharmaceutical processes and applications. Many sequence detection systems use multiple types of detectors and multiple types of signals output by the detectors to identify the entities and object types in the input target sequence. Often, electromagnetic sensors are used to generate electrical signals that vary depending on the dimensions and electrical properties of the object or entity passing through the electromagnetic sensor. Certain sequence detection systems use optical sensors to identify and classify entities and objects in the target sequence by the wavelengths of light absorbed by the object or entity, or by computational processing of optical images generated from the object or entity. Further detectors include weight detectors, volume detectors, and magnetic susceptibility detectors.

[0002] Sequence detectors are often characterized by the reliability and accuracy with which they determine and report the sequence of the input target object or entity, by the speed with which they identify the input target sequence, and by the cost and complexity of the sequence detector. Often, increased reliability and accuracy comes with increased cost and complexity. To optimally use sequence detectors in real-world applications, system and process engineers seek to evaluate and compare different types of sequence detectors for use in a particular application. For this reason, system and process engineers, researchers, diagnosticians, and other users of sequence detectors continually search for new and different types of sequence detectors to facilitate the identification and deployment of specific sequence detection processes and systems that optimally meet the sequence detection parameters and goals of a particular application. Summary of the Invention

[0003] overview The present specification discusses a detection system that includes a mechanical change sensor that exhibits one or more mechanical changes when it specifically interacts with an entity in a target. Each entity has a type, a mechanical change-to-signal converter that converts the one or more mechanical changes into a signal, and an analysis subsystem that uses the signal to determine the type of entity in the target. The present specification further discusses a sequence detection system that includes a nucleic acid-polymerase mechanical change component that exhibits a mechanical change when it specifically associates with a nucleotide polyphosphate in an active site; a mechanical change-to-signal converter that converts the mechanical change in the nucleic acid-polymerase mechanical change component into an output signal, and an analysis subsystem that uses an external signal to determine the sequence of the type of monomer in the nucleic acid polymer target. The present specification further discusses a method of determining the monomer sequence from the signal output by the sequence detection system, the method including: identifying for each signal portion a portion of the signal that each corresponds to a different monomer in the sequence of monomers; calculating n derived values ​​(n is an integer equal to or greater than 2) from the signal portion; mapping the n derived values ​​to an n-dimensional range volume that corresponds to a particular type of monomer; and generating and storing a symbolic representation of the sequence of the type of monomer that is complementary to the type of monomer assigned to the signal portion. In certain cases, the signal derivatives may be sufficient to identify different monomers in a sequence of monomers. [Brief description of the drawings]

[0004] [Figure 1] FIG. 1 illustrates, in one example, a target input to a first type of mechanical variation-based sequence detector and the resulting determined sequence-indicating output.

[0005] [Diagram 2] FIG. 2 shows one implementation of a sequence detection system based on the first type of mechanical variation-based sequence detector.

[0006] [Figure 3A-3F] 3A-3F show, in one example, the mechanical change sensor component of the sequence detection system shown in FIG.

[0007] [Figure 4] FIG. 4 illustrates, in one example, the generation of a voltage signal from a change in shape of a mechanical change sensor component by a mechanical change-detection subsystem in the array detection system shown in FIG.

[0008] [Diagram 5] FIG. 5 illustrates, in one example, the internal components of the mechanical variation-based sequence detector 204 shown in FIG.

[0009] [Figures 6A-6D] 6A-6D show, in one example, voltage signals generated by each of four different types of objects occurring within a target.

[0010] [Figure 7] FIG. 7 shows the virtual assay results generated by the sequence detection system from a test target containing 10 objects of each of four object types a, b, c, and d.

[0011] [Figure 8] FIG. 8 shows a plot of the average voltage-magnitude data contained in the data table shown in FIG.

[0012] [Figure 9] FIG. 9 shows a two-dimensional plot of the data contained in the table shown in FIG.

[0013] [Figure 10] FIG. 10 shows, in one example, the use of three derived values ​​to determine the object type.

[0014] [Figure 11]FIG. 11 summarizes the sequence detection system discussed above with reference to FIGS.

[0015] [Figure 12] FIG. 12 provides a table comparing one example first sequencing system discussed above with reference to FIGS. 1-11, and a second sequencing system discussed in this subsection of the specification.

[0016] [Figure 13] FIG. 13 illustrates, in one example, a mechanical change-based sequence detector included in the second sequence detection system.

[0017] [Figure 14A] 14A-14B show two different current-to-voltage converter circuits that may be used separately or together in various embodiments of the second sequence detection system.

[0018] [Figure 14B] 14A-14B show two different current-to-voltage converter circuits that may be used separately or together in various embodiments of the second sequence detection system.

[0019] [Figure 15] FIG. 15 shows, in one example, an array of cells that provides parallel sequencing.

[0020] [Figure 16] FIG. 16 shows a short DNA polymer.

[0021] [Figure 17A] 17A-17B show hydrogen bonds between purine and pyrimidine bases of two opposing parallel DNA strands.

[0022] [Figure 17B] 17A-17B show hydrogen bonds between purine and pyrimidine bases of two opposing parallel DNA strands.

[0023] [Figure 18A-18B] 18A-18B show double-stranded DNA.

[0024] [Figure 19] Figure 19 shows the names and chemical structures of the 20 common amino acids.

[0025] [Figure 20] Figure 20 shows the polymerization reaction catalyzed by the Klenow fragment of E. coli DNA polymerase I used as the mechanically altered sensor component in the second sequence detection system.

[0026] [Figure 21A] 21A-21E show copy strand elongation catalyzed by the Klenow fragment of E. coli DNA polymerase I.

[0027] [Figure 21B] 21A-21E show copy strand elongation catalyzed by the Klenow fragment of E. coli DNA polymerase I.

[0028] [Figure 21C] 21A-21E show copy strand elongation catalyzed by the Klenow fragment of E. coli DNA polymerase I.

[0029] [Figure 21D] 21A-21E show copy strand elongation catalyzed by the Klenow fragment of E. coli DNA polymerase I.

[0030] [Figure 21E] 21A-21E show copy strand elongation catalyzed by the Klenow fragment of E. coli DNA polymerase I.

[0031] [Fig. 22A-22B]22A-22B show, in one example, variable resistance components of a second array detection system.

[0032] [Figure 23A] 23A-23B show one method for attaching a DNA polymer tether to a DNA polymerase mechanical change sensor component 1304. FIG.

[0033] [Figure 23B] 23A-23B show one method for attaching a DNA polymer tether to a DNA polymerase mechanical change sensor component 1304. FIG.

[0034] [Figure 24] FIG. 24 illustrates some of the features of the DNA polymer tether that, in one example, mechanically couples a DNA polymerase to a variable resistance component and also forms part of the variable resistance component.

[0035] [Diagram 25] FIG. 25 shows numerous examples of four deoxynucleotide-monomer sequences with high resistance at low current and low resistance at high current.

[0036] [Figure 26A] 26A-26B show, in one example, tailoring the responsiveness of mechanical-to-electrical signal transduction via DNA polymer tether sequence design.

[0037] [Figure 26B] 26A-26B show, in one example, tailoring the responsiveness of mechanical-to-electrical signal transduction via DNA polymer tether sequence design.

[0038] [Figure 27] FIG. 27 shows, in one example, a DNA polymer tether featuring a DNA repeat deoxynucleotide sequence.

[0039] [Figures 28A-28C] 28A-28C show, in one example, an approach used in certain embodiments of the second sequence detection system to distinguish different deoxynucleotides added to the copy strand by a DNA polymerase.

[0040] [Figure 29A] Figures 29A-29C show, in one example, the use of locking oligonucleotides to securely hold a DNA polymer tether within a porin channel.

[0041] [Figure 29B] Figures 29A-29C show, in one example, the use of locking oligonucleotides to securely hold a DNA polymer tether within a porin channel.

[0042] [Figure 29C] Figures 29A-29C show, in one example, the use of locking oligonucleotides to securely hold a DNA polymer tether within a porin channel.

[0043] [Diagram 30] FIG. 30 shows the apply-voltage cycles used in one embodiment to prevent the appearance of disruptive noise in the output voltage signal. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0044] Discussed herein are sequence detectors that generate signals that can determine the sequence of entity types in a target. The sequence detectors described herein may be, for example, electromechanical devices. An electromechanical device is a device that includes both electrical and mechanical components, and may include additional optical, fluidic, and other components. An example sequence detector detects sequence by generating signals that can determine the sequence of entity types in a target. The sequence detector may include components, such as a microprocessor-controlled signal analysis component, that analyzes the signal to determine the sequence of entity types. A target includes an array of entities, each entity having a type. The sequence detector physically interacts with the target and generates a signal that changes when a mechanical change sensor in the sequence detector specifically interacts with a different type of entity in the target. A specific interaction is an interaction between the mechanical change sensor and an entity that deterministically generates a mechanical change in a mechanical change sensor characteristic of the entity type to which the entity belongs that is converted by a mechanical change-to-signal converter into a corresponding signal, which is also a characteristic of the entity type to which the entity belongs. The mechanical changes may include changes in the shape and / or size of the mechanical change-signal converter, changes in the position of the mechanical change-signal converter relative to other components, changes in the orientation of the mechanical change-signal converter relative to other components, and other such mechanical changes. Two different examples of targets and corresponding array detectors are discussed herein in the following two subsections. The target in the first example is a linear array of macro-scale objects connected together with a string or wire. Each object has one of four different shapes. The type of object corresponds to the shape of the object. The first corresponding array detector generates a time-varying electrical signal as the target passes the mechanical change sensor, which interacts specifically with each object, producing a mechanical change in the mechanical change sensor that is converted into an electrical signal characteristic of the object's shape by a variable resistor.The second example target is a biopolymer that contains a sequence of monomers that are covalently linked to each other. In DNA and RNA polymers, which are used as examples in the following description, there are four commonly occurring different types of monomers that differ from each other in chemical composition and structure, with additional types of monomers occurring less frequently. The second corresponding sequence detector generates a time-varying electrical signal when the target biopolymer passes through a mechanical change sensor, which specifically associates with the monomers to produce a mechanical change in the mechanical change sensor, which is then communicated to a variable resistor by a coupler. These two types of detectors are examples, and other configurations exist and can be implemented. First Type of Mechanical Change-Based Electromechanical Alignment Detector

[0045] FIG. 1 illustrates, in one example, a target input to a first (type) mechanical variation based sequence detector, and a determined sequence representation output. The target 102 is a linear array of objects 104-116. In this example, there are four different types of objects contained in the target: (1) a cylindrical object type 120, referred to as type a; (2) a cubic object type 122, referred to as type b; (3) a spherical object type 124, referred to as type c; and (4) a pyramidal object type 126, referred to as type d. The target 102 includes a linear spacing member 130, such as a wire or cord, to which the objects 104-116 are securely attached. The target is mechanically input to a first type sequence detector, which outputs a symbolic representation of the sequence of object types 132 within the target.

[0046] FIG. 2 illustrates one embodiment of a sequence detection system based on a first type of mechanical variation-based sequence detector. The sequence detection system 202 includes a mechanical variation-based sequence detector 204 to which a target 206 is mechanically input. The mechanical variation-based sequence detector mechanically outputs the target 208 and outputs an electrical signal 210 to an analysis subsystem. The analysis subsystem is implemented as a computer program executed on a particular sequence detector's computer system 212, processes the electrical signal to determine a sequence of object types in the target, and outputs the determined sequence of object types 214 on a computer display device 216. In another embodiment, the analysis subsystem is implemented by a processor-controlled subsystem other than a general-purpose computer system. The computer system may further store an encoded representation of the sequence in one or more memories and / or one or more mass storage devices. The encoded representation of the sequence may be transmitted to a remote computer system and then retrieved for display to a user and further analysis.

[0047] 3A-3F show, in one example, the mechanical change sensor component of the array detection system shown in FIG. 2. As shown in FIG. 3A, the mechanical change sensor component 302 is a funnel-shaped device that includes a rigid circular ring 304 to which a number of flexible spring-like tines, including tine 306, are attached. The tines are arranged as if on the surface of a cone with an inward radial orientation, as shown in the top-down projection of FIG. 3B. FIG. 3C shows a logical representation of the mechanical change sensor component 310. In a series of figures including FIGS. 3D-3F, the operation of the mechanical change sensor component is shown. An object 312 of type b (122 in FIG. 1) is shown positioned behind the mechanical change sensor component 302 of FIG. 3D. In FIGS. 3E-3F, the object is mechanically translated through the mechanical change sensor component 302. As it passes through the mechanical change sensor component, the object pushes the flexible tines outward, distorting the funnel shape of the mechanical change component, as shown in FIG. 3F.

[0048] FIG. 4 illustrates, in one example, the generation of a voltage signal from a change in the shape of a mechanical change sensor component by a mechanical change detection subsystem in the array detection system shown in FIG. 2. On the left hand side of FIG. 4, the mechanical change detection subsystem 402 is shown when the funnel-like shape of the mechanical change sensor component is not distorted. On the right hand side of FIG. 4, the mechanical change detection subsystem 404 is shown when the funnel-like shape of the mechanical change sensor component is distorted by the presence of an object within the mechanical change sensor component. The mechanical change detection subsystem 402 includes the mechanical change sensor component 302, a potentiometer 406, and a voltmeter 408. The mechanical change sensor component 302 is mechanically connected to the potentiometer 406 by a cord or wire 410 that is attached at one end to one of the tines 412 of the mechanical change sensor component and at the other end to a slidable potentiometer arm 414. The cord or wire 410 passes through three freely rotating pulleys 416-418. The slidable potentiometer arm 414 is held in a first position by the tines 412 against the force of a weak spring 420 in the potentiometer arm cylinder 422. As shown on the right hand side of FIG. 4, when the mechanical change sensor component is distorted by the presence of an object, the tines 412 are pushed downwards, which in turn causes the slidable potentiometer arm 414 to be pulled upwards by the spring 420 in the potentiometer arm cylinder 422. In this example, the potentiometer 406 functions as a variable resistor. A variable resistor is a circuit element with a resistance to current flow that can be altered, and a potentiometer is one example of a variable resistor. In the position shown on the left hand side of FIG. 4, the potentiometer arm is connected to the potentiometer circuit 424 below the resistor 426, which results in little or no voltage drop across the voltmeter 408. However, when the potentiometer arm is in the position shown on the right hand side of FIG. 4, the potentiometer arm is connected to the potentiometer circuit 424 at a point midway through resistor 428, and as a result there is a significant voltage drop across the voltmeter.Thus, the mechanical change-detection subsystem generates a voltage signal that varies in response to the degree of geometric distortion of the mechanical change sensor component, with the magnitude of the output voltage signal corresponding to the degree of distortion of the mechanical change sensor component.

[0049] Figure 5 illustrates, in one example, the internal components of the mechanical change based sequence detector 204 shown in Figure 2. The mechanical change based sequence detector includes the mechanical change-detection subsystem 402 shown in Figure 4 and a pair of two electric motor driven counter-rotating gear drums 502 and 504 that feed a target 506 object through the mechanical change sensor component 302. The pair of counter-rotating gear drums feeds the target 506 object through the mechanical change sensor component 302. As the target is pulled through the mechanical change sensor component, a voltmeter outputs a voltage signal 210 that is input to the analysis subsystem (212 in Figure 2).

[0050] 6A-6D show, in one example, voltage signals generated by each of four types of objects occurring within a target. FIG. 6A shows a plot 602 of the voltage signal generated when a type d object 604 passes through the mechanical change sensor component. The voltage of the output signal is represented by the vertical axis 606, and the position of the object in the horizontal direction is represented by the horizontal axis 608 of the plot. Note that the position may be expressed in either horizontal displacement or time, assuming the target moves through the sensor at a constant speed. Because type d objects are rotationally unstable about an internal axis passing through the center of the top apex and base, type d objects tend to rotate back and forth about this axis when passing through the mechanical change sensor component. They also tend to rotate about the four internal horizontal axes. As a result, the voltage signal 610 tends to oscillate as the object passes through the mechanical change sensor. A type c object 612 produces a smooth, symmetrical signal 614, as shown in FIG. 6B. A type b object 616 shows a symmetrical signal 618 with slight oscillations due to slight rotational instability, as shown in FIG. 6C. Object 620 of type a also produces a symmetric output signal 622 with slight oscillations, as shown in FIG. 6D. The output voltage signals are analyzed by the computational analysis subsystem (212 in FIG. 2). Data is collected from the region of each voltage-signal curve that begins when the voltage signal rises to half its peak height and ends when the signal falls back to half its peak height, as shown in each plot in FIGS. 6A-6D by the arrows, such as arrow 624, and vertical dashed lines, such as vertical dashed lines 626 and 628. The analysis subsystem calculates the voltage-signal curves for each object, the mean voltage magnitude μ, such as mean voltage magnitude 630, and the variance σ, such as variance 632. 2 , and the area A under the voltage-signal curve, such as area 634.

[0051] The calculated value is the voltage amplitude v of n samples taken from different time points or displacements within the central portion of the voltage-signal curve. iIn various different embodiments, the current magnitude or other values ​​may be sampled instead. For example, a sampling frequency of 1 KHz would result in 1000 sample voltage magnitudes. The area A is calculated by the discrete integral:

number

number

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[0052] FIG. 7 shows hypothetical analysis results generated by the array detection system from a test target containing 10 objects of each of four object types a, b, c, and d. The analysis results for each of the four object types are shown in Tables 702-705. Each table includes three columns corresponding to the calculated voltage-magnitude mean, variance, and area of ​​each voltage-signal curve output by the mechanical change based array detector. As is often found in experimental data, the three calculated values ​​vary across the ten examples of the four different object types. The analysis subsystem uses the output voltage-signal curves to attempt to uniquely identify the type of each object that passes through the mechanical change sensor component.

[0053] A common way of using the output voltage signal is to select a single calculated value, such as the average voltage magnitude, to distinguish each type of object from the remaining types of objects. FIG. 8 shows a plot of the average voltage-magnitude data contained in the data table shown in FIG. 7. A key 802 is shown in the upper left-hand portion of FIG. 8. The key describes the various symbols used to plot the average voltage magnitude values ​​for each of the various types of objects. The lower portion of FIG. 8 shows a plot 804 of the average voltage magnitude data against a horizontal axis 806 representing voltage magnitude. The average voltage amplitude values ​​for each type of object are clustered within a sub-region of the horizontal axis as shown in FIG. 8 by dashed ellipses 808-811. The average voltage magnitude of objects of type c, for example, falls within the voltage-magnitude range indicated by ellipse 808. From the data plot shown in FIG. 8, it can be seen that type c objects can be uniquely distinguished from the remaining object types based solely on the average voltage magnitude, since in this example there is no overlap between the range of average voltage magnitudes of objects of type c and the range of average voltage magnitudes of objects of types a, b, and d. Similarly, objects of type b, whose average voltage magnitude values ​​fall within the range represented by oval 811, are uniquely distinguishable from the remaining types of objects based on their average voltage magnitude alone. However, the average voltage magnitude ranges of objects of types d and a, represented by ovals 809-810, overlap almost completely with one another, and as a result, objects of types d and a cannot be distinguished using their average voltage magnitude values ​​alone.

[0054] In some instances, if a particular output signal is insufficient to distinguish between different types of objects in an array, the array detector designer seeks to incorporate additional types of sensors into the array detector to generate additional output signals such that the combination of the multiple output signals provides sufficient information to distinguish the objects from one another. For example, a change counter uses separate size-detection sensors, weight detectors, and magnetic-susceptibility detectors to generate separate output signals that together provide distinct output signal signatures for each type of coin. In contrast, the array detection system of the examples described herein calculates multiple derivatives from a single output voltage signal to distinguish each type of object in the target. As discussed above with reference to Figures 6A-6D and 7, the analysis subsystem calculates not only the average voltage magnitude but also the variance from the voltage-signal curves corresponding to the objects. Figure 9 shows a two-dimensional plot of the data contained in the table shown in Figure 7. The horizontal axis 902 represents the average voltage magnitude obtained from the voltage-signal curves as in Figure 8, and the vertical axis 904 represents the variance obtained from the voltage-signal curves. As in FIG. 8, dashed ellipses 906-909 enclose clusters of data points plotted for different object types. As can be easily seen in FIG. 9, the two-dimensional areas contained within these ellipses do not overlap. Thus, the mean voltage magnitude and variance pair calculated from a single output voltage signal for a particular object contains enough information to unambiguously assign a type to the object. In other words, in this example, the single output voltage signal generated by the mechanical change-detection subsystem contains enough information to assign a type to each object, but the information within each voltage-signal curve is, in a sense, two-dimensional.

[0055] As discussed with reference to FIG. 9, the use of two derived values ​​including the magnitude and variance of the mean voltage by the analysis subsystem of the array detection system is sufficient to identify each object or entity within the target as described with reference to FIG. 1. The analysis system uses the two derived values ​​as coordinates to map the two derived values ​​to range areas corresponding to specific object or entity types. If the range areas overlap, additional derived values ​​such as calculated areas under the voltage-signal curve may be used to uniquely distinguish the types of objects within the target. FIG. 10 illustrates the use of three derived values ​​in determining object type in one example. In FIG. 10, each of the three different derived values ​​is represented by three axes 1002, 1004, and 1006. The plotted data points for the four different object types fall into four separate non-overlapping elliptical range volumes 1010-1013. In general, an increase in the number of derived values ​​reduces the probability of overlap in the range of derived values ​​for different object types when the derived signals are reasonably orthogonal and sensitive to differences in object types.

[0056] FIG. 11 summarizes the sequence detection system discussed above with reference to FIGS. 1-10. A target, including a sequence 1102 of objects or entities, is input to a sequence detection system 1104 and mechanically translated by a machine translation component 1108 via a mechanical change component 1106. A power source 1110 provides power for the machine translation. The mechanical change component 1106 is mechanically coupled to a variable resistance component 1114 by a coupler 1112. A mechanical coupler couples two or more entities together by a physical coupler, such as a string or cord, in macroscale devices, or a linear molecule, such as a DNA polymer, in nanoscale and microscale devices. The variable resistance component 1114 provides a variable resistance to a current 1116 in response to a movement of the coupler 1112 caused by a change in the shape of the mechanical change component 1106. A power source 1118 drives the current flow 1116. The measurement component 1120 measures the electrical potential of the current flow channels or the current flow itself to generate an output electrical signal 1122 that changes with the change in geometry of the mechanical change component 1106. The output signal is computationally processed by an analysis subsystem, not shown in Figure 11, to generate an indication of the arrangement of object types in the target 1102. The arrangement detection systems discussed above with reference to Figures 1-11 are macro-scale devices that determine the arrangement of macro-scale object types within the target. Second Type of Mechanical Change-Based Electromechanical Array Detector

[0057] The second (type) sequence detection system discussed herein is a mixed-scale device that includes macroscale, microscale, and nanoscale components. The second sequence detection system determines the sequence of deoxynucleotide monomers within a nucleic acid polymer.

[0058] FIG. 12 is a table comparing the first sequence detection system discussed above with reference to FIGS. 1-11 and the second sequence detection system discussed in this subsection of the specification. The first column 1202 of the table lists the sequence detection system components discussed above with reference to FIG. 11. The second column 1204 of the table further describes each component listed in the first column with respect to the first sequence detection system. The third column 1206 further describes each component listed in the first column with respect to the second sequence detection system, which are further described below. In the first sequence detection system, the power source for the machine translation of the target is current available from a battery or line current, whereas in the second sequence detection system, the power source for the machine translation of the target is chemical energy generated by hydrolysis of phosphoanhydride bonds and hydrolysis of inorganic pyrophosphates. In the first sequence detection system, the machine translator that translates the target is two pairs of counter-rotating electric motor driven gear drums, as shown in row 1210 of Table 2, and in the second sequence detection system, the machine translator is the Klenow fragment of E. coli DNA polymerase, as shown in row 1212 of the table. In the first sequence detection system, the mechanically variable component is a series of funnel-like springs or bristles, and in the second sequence detection system, the mechanically variable component is the Klenow fragment of DNA polymerase I, as shown in row 3 of the table, 1212. In the first sequence detection system, the coupler is a cord or wire, and in the second sequence detection system, the coupler is a DNA polymer, as shown in row 4 of the table, 1214. In the first sequence detection system, the variable resistor is a potentiometer, and in the second sequence detection system, the variable resistor is a portion of a DNA polymer coupler present in the MspA-porin channel, as shown in row 5 of the table, 1216. In both sequence detection systems, it is an electric current that flows. In the first array detection system, the charge carriers are conduction band electrons flowing in metal wires, and in the second array detection system, the charge carriers are positively and negatively charged ions, as shown in the sixth row of the table, 1218. But, of course, the electrodes are connected by current carrying wires.In the first array detection system, the power source driving the current is derived from a battery or line current, and in the second array detection system, the power source driving the current is derived from the line current, as shown in the seventh row of the table 1220. In the first array detection system, the current or potential measuring device is a voltmeter, and in the second array detection system, the current or voltage measuring device is a current-to-voltage converter, as shown in the eighth row of the table 1222. Thus, the first and second array detection systems are similar to each other in configuration and operation but include different specific types of components.

[0059] FIG. 13 illustrates, in one example, a mechanical change-based sequence detector included in a second sequence detection system. In the following discussion, the mechanical change-based sequence detector 1302 is referred to as a "cell." The diagram in FIG. 13 does not reflect the relative sizes and volumes of the various components. In many embodiments, the cell 1302 is a macroscale or microscale device, while the mechanical change sensor component 1304 is a nanoscale component. FIG. 13 is intended to illustrate the overall configuration and relative positions and orientations of the various components of the mechanical change-based sequence detector, rather than accurately depicting the relative scale of the components.

[0060] The cell 1302 includes a two-part container 1306 with a first solution-containing chamber 1308 separated from a second solution-containing chamber 1310 by a Teflon barrier 1312 and a lipid bilayer 1314. In one embodiment, the lipid bilayer comprises 1,2-diphytanoyl-sn-glycerol-3-phosphocholine. The Teflon barrier includes an opening 1316 that is covered with a lipid bilayer, such that the first solution-containing chamber 1308 and the second solution-containing chamber 1310 are separated only by the lipid bilayer in the opening 1316. A narrow channel 1316 through the lipid bilayer is provided by a mycobacterial porin ("MspA porin"), an octameric protein aggregate with eight-fold symmetry. The narrow channel is wide enough to allow passive diffusion of ions between the two solution-containing chambers. The first and second solution-containing chambers 1308 and 1310 contain a buffer solution at pH 8.0. In one embodiment, the buffer comprises 50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid ("HEPES"), 150 mM KCl, 1 mM dithiothreitol ("DDT"), and 3 mM MgCl2. HEPES is a zwitterionic buffer compound. KCl provides ions that carry the ionic current. Dithiothreitol is a reducing agent that promotes free sulfhydryl groups on proteins. MgCl2 provides MgCl2, which aids in the catalytic activity of the mechanical change sensor component 1304, discussed below. 2+ions. The first solution-containing chamber 1308 further includes deoxynucleotide triphosphates and primer-associated deoxyribonucleic acid templates. The primer-associated deoxyribonucleic acid templates 1318-1319 are targets for which the sequence of deoxynucleotide monomers is determined by a second sequence detection system. In certain embodiments, the second solution-containing chamber 1310 further includes a lock component, discussed below. The solution in the second solution-containing chamber 1310 is in fluid contact with a positive or reference electrode 1320, and the solution in the first solution-containing chamber 1308 is in fluid contact with a negative electrode 1322. In one embodiment, a silver / silver chloride electrode is used. When a voltage is applied to the cell through the electrodes, negative ions flow through the porin channel toward the positive anode, and positive ions flow through the porin channel toward the negative electrode. In one example, if DNA or RNA polymers are present within the pores, they may inhibit the downward flow of negative ions. In other words, a current is established in the porin channel by applying a voltage to the two electrodes 1320 and 1322. At various times during operation of the cell, the polarity of the applied voltage can be temporarily reversed by reversing the polarity of the electrodes, as discussed further below.

[0061] In another embodiment, rather than using a Teflon barrier and lipid bilayer, a photolithography process is used to create openings in a silicon substrate or other type of substrate, and a synthetic polymer membrane is used to prevent fluid communication between the two chambers other than through the porin channel. In one example, a triblock copolymer may be used for the membrane. In another embodiment, MspA-porin variants may be used, including single-chain versions or versions containing fewer or more than eight subunits. Certain variants may contain multiple subunits that differ in sequence. Other types of pore-containing biopolymers and synthetic polymers may alternatively be used in another embodiment. In another embodiment, Mn 2+ Additional types of divalent metal ions may also be used, including Ca. 2+ and Sr 2+Non-catalytic metal ions may also be used, including:

[0062] In one embodiment, the mechanical change sensor component 1304 is the Klenow fragment of E. coli DNA polymerase I. The Klenow fragment is obtained by removing the 5'→3' exonuclease structural domain from E. coli DNA polymerase I by treating it with a protease or by expressing the fragment of interest from a genetically engineered bacterial strain. The Klenow fragment retains the 5'→3' polymerization function. As described further below, when supplied with a primer-associated DNA template and deoxynucleotide triphosphates, the Klenow fragment of E. coli DNA polymerase I catalyzes the stepwise polymerization of deoxynucleotide triphosphates to form a copy DNA strand complementary to the sequence of the template DNA strand. The binding component 1324 is a DNA polymer tether. It attaches to the mechanical change sensor component 1304 and is drawn into the porin channel by the voltage applied to the electrode, since the DNA polymer is negatively charged and moves toward the positive electrode under an applied voltage. As discussed further below, the small region of the DNA polymer tether that spans the narrow constriction in the porin channel acts together as a variable resistor that regulates the flow of ions along the narrow constriction between the two solution-containing chambers to different degrees depending on the position of the small region of the DNA-polymer tether relative to the narrow constriction. Because a system containing the Klenow fragment of E. coli DNA polymerase I and a porin exhibits differences for each different type of deoxynucleotide triphosphate that occupies the active site in the Klenow fragment of E. coli DNA polymerase I, the DNA-polymer tether has a different dynamic position relative to the narrow constriction in the porin channel when a different deoxynucleotide triphosphate specifically associates with the active site, which is reflected in a different dynamic current through the porin channel for each different type of deoxynucleotide triphosphate that is sequentially incorporated into the growing DNA polymer strand.Specific association between a molecule and an active site includes key-in-lock or induced-fit type associations, where certain electrostatic and chemical features of a molecule, in association with an active site of complementary electrostatic and chemical features, lead to greater binding affinity for a molecule or class of molecules than for molecules that do not specifically associate with the active site. The differences can be due to one or more conformational changes, movement of the Klenow fragment relative to the porin channel, and other changes. The current detection circuitry discussed below generates a voltage signal that varies in response to changes in current through the porin channel. In alternative embodiments, in place of the Klenow fragment described above, many different polymerases, polymerase fragments, and other types of biomolecules that interact with biopolymer targets for sequencing can be used. Many types of natural and synthetic nucleotides can be used, including larger phosphate esters such as deoxynucleotide hexaphosphates, nucleotides with different carbohydrate moieties, different bases, and different functional groups. Many additional types of mechanically altered components can be used to sequence a wide variety of target biopolymers and synthetic polymers. This includes enzymes and other proteins, and protein / nucleic acid complexes that interact with target proteins in a sequence-specific manner. Also note that the phrase "mechanically altered sensor component" as applied to a second sequence detection system indicates that, as an example, signal generation is indicative of a change in shape of the polymerase fragment, a change in the relative position of the polymerase fragment with respect to the porin, and / or a change in the orientation of the polymerase fragment with respect to the porin. In certain embodiments, the change in shape of the polymerase fragment provides a mechanical change that leads to movement of the variable resistor. Ultimately, the mechanical altered sensor component and its interaction with the target and pore-containing components results in a mechanical change in the position of the variable resistor. In another embodiment, coupling connectors and variable resistors other than DNA polymers can be used.

[0063] The second sequence detection system is similar to the first sequence detection system, although the first sequence detection system is a macro-scale system and the second sequence detection system is a mixed-scale system including macro-scale and nano-scale components. Both sequence detection systems use a mechanical change sensor component to generate a mechanical signal that varies with the type of object or entity currently being processed or associated with by the mechanical change sensor component. Both sequence detection systems employ a mechanical change sensor component to generate a mechanical signal that varies with the type of current object or entity being processed by or associated with the mechanical change sensor component. Both sequence detection systems employ a mechanical coupling to couple the mechanical change sensor component to a variable resistance component. Both sequence detection systems generate an output voltage signal by converting the mechanical signal generated by the mechanical change sensor component to an electrical signal. Both sequence detection systems use computational analysis of the output signal to generate a plurality of derived values ​​that are used together to identify the sequence of object or entity types in the target sequence. In the second sequence detection system, the detected deoxynucleotide-monomer type sequences are complementary to and have opposite polarity to the deoxynucleotide-monomer type sequences in the template strand target.

[0064] Figures 14A-14B show two different current-to-voltage converter circuits that may be used separately or together in various embodiments of the second array detection system. In Figure 14A, a voltage source 1402 applies a voltage to a cell 1404. An inverting amplifier, or op-amp 1406, with a feedback loop 1408 that includes a feedback resistor 1410, outputs a voltage signal proportional to the current through the cell. A second op-amp 1412 amplifies the voltage difference of the inputs to produce an amplified voltage signal that is passed through a frequency correction circuit 1414 to produce a final output voltage signal 1416 proportional to the current through the cell 1404. Figure 14B shows a current-to-voltage converter that uses a feedback capacitor rather than a feedback resistor.

[0065] FIG. 15 illustrates, in one example, an array of cells that provides parallel sequencing. Parallel sequencing can be used to increase the sequencing throughput of a system. The array of cells includes multiple cells, such as cell 1502, and an analysis subsystem 1504 that processes and analyzes the voltage signal output from the multiple cells in parallel. In general, the sequences output by the sequence detection system may include errors due to a variety of different operational error sources that are often present in the sequence detection system components, including mechanically changed sensor components, mechanically coupled variable resistance components, and current or potential measurement circuitry. Thus, depending on the level of accuracy desired, multiple identical targets can be sequenced and a consensus sequence can be computationally generated from the multiple sequences determined for the multiple identical targets. In many cases, sequencing of multiple targets is desirable. In the array of cells illustrated in FIG. 15, each cell may be loaded with multiple copies of each of multiple different types of targets. The cells are processed in a non-deterministic order based on random association of the Klenow fragment of E. coli DNA polymerase I with the primer-associated template to sequentially generate sequence information from the multiple types of targets. The analysis subsystem 1502 serially collects sequence information from multiple cells, such as cell 1504, assigns each sequence to a group of sequences generated by a particular target, and then assembles a consensus sequence for each different type of target from the group of sequences generated from each target type. The use of parallel processing allows for rapid and efficient consensus sequencing of multiple targets.

[0066] Figures 16-19 show deoxyribonucleic acids and peptides. Figure 16 shows a short DNA polymer. Deoxyribonucleic acid ("DNA") is a linear polymer synthesized from four different deoxynucleotide triphosphates, which when incorporated into the polymer are called deoxynucleotide monomers. Deoxynucleotide monomers include: (1) deoxyadenylate, abbreviated "A", a purine-containing deoxynucleotide; (2) deoxythymidylate, abbreviated "T", a pyrimidine-containing deoxynucleotide; (3) deoxycytidylate, abbreviated "C", a pyrimidine-containing deoxynucleotide; and (4) deoxyguanidylate, abbreviated "G", a purine-containing deoxynucleotide. The corresponding nucleosides lack the phosphate group attached to the ribose hydroxyl oxygen via a phosphodiester bond and are called deoxyadenosine, deoxythymidine, deoxycytidine, and deoxyguanosine. FIG. 16 shows a short DNA polymer 1600, called an "oligomer" or "oligonucleotide," which consists of the following subunits: (1) deoxyadenylic acid 1602; (2) deoxythymidylic acid 1604; (3) deoxycytidylic acid 1606; and (4) deoxyguanidylic acid 1608. The deoxynucleotide subunits are linked together via phosphodiester bonds 1610-1615 to form a DNA polymer. A linear DNA molecule, such as the oligomer shown in FIG. 16, has a 5' end 1618 and a 3' end 1620. Often, the 5' end 1618 contains a phosphate group attached to the 5' hydroxyl oxygen via a phosphoester bond. A DNA polymer can be chemically characterized by writing the one-letter abbreviations of the deoxynucleotide subunits that together make up the DNA polymer in sequence from the 5' end to the 3' end. For example, the oligomer 1600 shown in FIG. 16 can be symbolically represented as "ATCG." Deoxynucleotides contain a purine or pyrimidine base (e.g., adenine 1622 of deoxyadenylate 1602), a deoxyribose sugar (e.g., deoxyribose 1624 of deoxyadenylate 1602), and a phosphate group (e.g., phosphate 1626) that links one deoxynucleotide to another in a DNA polymer.Many non-natural nucleotides can be incorporated into DNA-like and RNA-like polynucleotides. Examples of modified nucleotides that can be included in a polynucleotide are inosine, xanthanine, hypoxanthanine, isocytosine, isoguanine, 2-aminopurine, 5-methylcytosine, 5-hydroxymethylcytosine, 2-aminoadenine, 6-methyladenine, 6-methylguanine, 2-propylguanine, 2-propyladenine, 2-thiolyracil, 2-thiothymine, 2-thiocytosine, 15-halouracil, 15-halocytosine, 5-propynyluracil, 5-propynylcytosine ... These include tosine, 6-azo uracil, 6-azo cytosine, 6-azo thymine, 5-uracil, 4-thio uracil, 8-halo adenine or guanine, 8-amino adenine or guanine, 8-thiol adenine or guanine, 8-thioalkyl adenine or guanine, 8-hydroxyl adenine or guanine, 5-halo substituted uracil or cytosine, 7-methyl guanine, 7-methyl adenine, 8-azaguanine, 8-azaadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, 3-deazaadenine, etc. Certain nucleotide analogs, such as adenosine 5'-phosphosulfate, cannot be incorporated into a polynucleotide. No nucleotide analogues can be incorporated into a polynucleotide.

[0067] DNA polymers containing the tissue information of an organism occur in pairs in the nucleus of a cell, forming a double-stranded DNA helix. One polymer of the pair is laid out in the 5' to 3' direction and pairs with a complementary polymer laid out in the 3' to 5' direction. The two DNA polymers of a double-stranded DNA helix are therefore described as antiparallel. The two DNA polymers, or strands, in a double-stranded DNA helix bind to each other through hydrophobic interactions between stacked purine and pyrimidine bases, and hydrogen bonds between purine and pyrimidine bases, an attraction accentuated by the conformational constraints of the DNA polymer. Due to many chemical and geometric constraints, a double-stranded DNA helix is ​​most stable when the deoxyadenylated subunits of one strand hydrogen bond to the deoxythymidylic acid subunits of the other strand, and the deoxyguanylic acid subunits of one strand hydrogen bond to the corresponding deoxycytidylic acid subunits of the other strand.

[0068] Figures 17A-17B show hydrogen bonds between purine and pyrimidine bases of two antiparallel DNA strands. Figure 17A shows hydrogen bonds between adenine and thymine bases of corresponding deoxyadenylate and deoxythymidylate subunits, and Figure 17B shows hydrogen bonds between guanine and cytosine bases of corresponding deoxyguanidylate and deoxycytidylate subunits. Note that the adenine / thymine base pair has two hydrogen bonds 1002 and 1003, while the guanosine / cytosine base pair has three hydrogen bonds 1004-1006. As a result, GC base pairs provide greater thermodynamic stability to the DNA duplex than AT base pairs. The AT and GC base pairs shown in Figures 10A-B are known as Watson-Crick ("WC") base pairs.

[0069] Figures 18A-18B show double-stranded DNA. As shown in Figure 18A, two strands of DNA polymer 1802 and 1804 with complementary sequences form an antiparallel double-stranded complex through hydrogen bonds between complementary bases in the two strands. The double-stranded complex is antiparallel because the two strands have opposite 5'-3' orientations or polarities. Adenine bases in one strand 1806 pair with thymine bases 1808 in the other strand, and guanine bases in one strand 1810 pair with cytosine bases 1812 in the other strand. The sequence of deoxynucleotides in the 5'-3' direction along one strand is complementary to the sequence of deoxynucleotides in the 3'-5' direction along the other strand. Complementarity of the two strands in an antiparallel double-stranded DNA polymer is generated when DNA polymerase catalyzes the polymerization of the copy strand onto the template strand. FIG. 18B shows the familiar double-stranded DNA conformations that occur under physiological temperature, pressure, pH, and ionic concentration.

[0070] Figure 19 shows the names and chemical structures of 20 common amino acids. Amino acids polymerize to form proteins in the ribosome-mediated translation process. Amino acids polymerize to form proteins in the ribosome-mediated translation process. Figure 19 shows a short four-amino acid polymer called a "peptide" 1902, which contains alanine, glutamic acid, glycine, and lysine monomers. Protein polymers typically have hundreds to thousands of amino acid monomers. Many proteins, such as MspA porin, contain multiple protein polymers. Under physiological conditions, proteins typically have complex three-dimensional conformations, such as the goblet-like conformation of the MspA porin octamer.

[0071] Figure 20 shows a polymerization reaction catalyzed by the Klenow fragment of E. coli DNA polymerase I used as the mechanical change sensor component in the second sequence detection system. This polymerization reaction adds a nucleotide triphosphate 2002 to the 3' end of the growing copy strand 2004. In Figure 20, the remaining deoxynucleotide monomers of the copy strand are indicated by an arrow 2006 and a 5' label 2008. The deprotonated 3' hydroxyl 2010 of the 3' terminal deoxynucleotide monomer of the copy strand performs a nucleophilic attack on the alpha phosphate 2012 of the deoxynucleotide triphosphate 2002, forming a phosphodiester bond 2014 and displacing an inorganic pyrophosphate 2016. This reaction, under standard physiological conditions, results in a relatively small free energy change, but is accompanied by a large free energy change caused by the subsequent hydrolysis of the pyrophosphate 2018. The chemical energy released by the hydrolysis of pyrophosphate not only drives the polymerization reaction but also drives the translation of DNA polymerase onto the primer-associated template strand, which may contribute to the different dynamic conformations exhibited by DNA polymerase when different deoxynucleotide triphosphates specifically associate with the active site and form hydrogen bonds with complementary deoxynucleotide monomers in the template strand.

[0072] Figures 21A-21E show copy strand extension catalyzed by the Klenow fragment of E. coli DNA polymerase I. Figures 21A-E all use the same drawing convention, which is now explained with reference to Figure 21A. The DNA polymerase is represented by a sphere 2102, which appears circular in cross section. The active site within the DNA polymerase is represented by a vertically shaded rectangle 2104. The template DNA strand 2106 and copy DNA strand 2108 are represented by a series of rectangles and disks. Purine and pyrimidine bases are represented by long vertically labeled rectangles, such as rectangle 2110, which represents a guanine base. The ribose moieties within each deoxynucleotide monomer are represented by small squares, such as small square 2112. The phosphodiester bonds that connect the deoxynucleotide monomers within the strand are represented by circles, such as circle 2114. Curved arrows, such as curved arrow 2116, indicate that the strand continues in the indicated direction.

[0073] FIG. 21A shows a DNA polymerase with no deoxynucleotide triphosphate occupying the active site. The DNA polymerase is ready to receive the next deoxynucleotide triphosphate for addition to the 3' end of the copy strand. The active site of a DNA polymerase is a complex chemical environment that includes several bound magnesium ions and numerous functional groups on the amino acid monomer side chains, all of which contribute to catalysis of the polymerization reaction, including specific binding of the template and copy strands, specific binding of deoxynucleotide triphosphates for addition to the 3' end of the copy strand via the reaction shown in FIG. 20, and stabilization of one or more transition states. In FIG. 21B, deoxynucleotide triphosphates diffuse through the channel of the DNA polymerase toward the active site. In at least one example, any of four different types of deoxynucleotide triphosphates may approach the active site, but only the deoxynucleotide triphosphate complementary to the unpaired deoxynucleotide monomer of the template strand in the active site will stably associate with the active site due to hydrogen bonding with the template strand deoxynucleotide monomer present in the unpaired active site, as shown in FIG. 21C. The stable association of deoxynucleotide triphosphates with the active site is associated with conformational changes in DNA polymerase, represented by the oval in FIG. 21C. The actual conformational changes are complex and affect several different domains within the DNA polymerase. The conformation of DNA polymerase is dynamic and is generally associated with various types of subtle vibrational modes and relative motions of various structural domains. The specific association of different types of deoxynucleotide triphosphates with the active site induces different dynamic DNA polymerase conformations, changes in the relative positions or orientations of the DNA polymerase and the porin, and / or other changes that are believed to be the source of mechanical signals of mechanical changes generated by the DNA polymerase that act as mechanical change sensor components of the second sequence detection system.

[0074] In the sequence of Figures 21C-21D, the polymerization reaction shown in Figure 20 occurs, forming a phosphodiester bond bridge 2120 incorporating the new deoxynucleotide monomer into the copy strand. Pyrophosphate 2122 is released from the active site. In the sequence of Figures 21C-21E, the DNA polymerase translates in association with the template and copy strand, re-forming an active site 2126 free of nucleotide triphosphates, ready to specifically incorporate a subsequent deoxynucleotide triphosphate. Note that the conformation of the DNA polymerase returns to its original conformation, represented in Figure 21E by a sphere 2128. Note that in certain embodiments, the specific association of the deoxynucleotide monomer with the active site, alone and without incorporation, can lead to a mechanical change in the polymerase that can be translated into a signal capable of determining the target sequence. In these embodiments, the nucleotide does not need to be incorporated into the copy strand.

[0075] 22A-22B show, in one example, the variable resistance component of a second sequence detection system. As shown in FIG. 22A, a DNA-polymer tether 2202 is attached to a DNA polymerase 2204 and extends through a porin channel to a second solution-containing chamber 1310 of the cell below a lipid bilayer 2206. Because deoxynucleotide triphosphates are specifically associated with the active site of the DNA polymerase during copy strand extension, dynamic conformational changes in the DNA polymerase result in translation of the DNA-polymer tether relative to the narrow conformation 2208 of the porin channel.

[0076] FIG. 22B shows that translation of a DNA-polymer tether with respect to a narrow constriction of the porin channel changes the resistance to ion flow through the porin channel. FIG. 22B shows four different positions of a DNA polymer tether within a porin channel 2210-2213. The DNA-polymer tether is represented as a series of circles of different diameters. Larger diameter circles, such as circle 2216, represent one or more deoxynucleotide monomers that, when positioned within the narrow constriction 2218 of the nascent channel, provide high resistance to ion flow through the porin channel. Circles of progressively smaller diameter represent one or more deoxynucleotide monomers that, when positioned within the narrow constriction, provide less and less resistance to ion flow through the porin channel. In a first position 2210, one or more low resistance deoxynucleotide monomers are positioned within the narrow constriction 2218, resulting in a relatively high percentage of ionic current through the porin channel. A number of positive 2220 and negative 2222 ion symbols are shown entering the porin channel. As the DNA polymer tether moves upward relative to the narrow constriction, one or more deoxynucleotide monomers that offer increasingly greater resistance to ion flow at positions 2211-2213 move into the narrow constriction, resulting in an increasingly smaller percentage of ion-current through the porin channel. Thus, positioning of the DNA polymer tether within the porin channel changes the resistance to ion flow through the porin channel, and the mechanical-change signal generated by the specific incorporation of deoxynucleotide triphosphates into the active side of the DNA polymerase is converted into an output voltage signal by the current-to-voltage conversion circuitry discussed above with reference to Figures 14A-14B. Details of the second type of mechanical change-based electromechanical array detector

[0077] FIG. 23A-23B shows one method for attaching a DNA polymer tether to a DNA polymerase mechanical change sensor component 1304. In wild type E. coli DNA polymerase I, a leucine monomer 2302 occupies amino acid monomer position 790. In FIG. 23A-23B a cysteine ​​monomer 2304 is used in place of the leucine monomer at position 790 by genetic engineering and biotechnology techniques to generate a mutant E. coli DNA polymerase I with a single free sulfhydryl moiety 2305, designated "P" in FIG. 23A-23B. This is the attachment site for the DNA polymer tether. Any of a variety of linker molecules, including linker molecule 2306, can be incorporated at one end of the DNA-polymer tether. In many embodiments, one or several deoxynucleotide monomers make up the 5' portion 2308 of the DNA-polymer tether and several dozen deoxynucleotide monomers make up the 3' portion 2310 of the DNA-polymer tether. A primary amine functional group 2312 provides the attachment point for the DNA polymer tether. The primary amine is attached to a bridging molecule 2314 containing a sulfhydryl-reactive maleimide moiety 2316 to generate an activated tether 2318, designated "L" in Figures 23A-23B. As shown in Figure 23B, a mutant E. coli DNA polymerase I with a single free sulfhydryl moiety 2305, P, and an activated tether 2318, L, reacts to covalently attach the tether to DNA polymerase 2320. The maleimide moiety 2316 of the activated tether reacts with the free sulfhydryl moiety 2304 of the mutant DNA polymerase to attach the DNA-polymer tether 2318 to the DNA polymerase 2320. This is just one example of many different possible ways to attach a DNA polymer tether to a DNA polymerase.

[0078] FIG. 24 illustrates several features of a DNA polymer tether that mechanically couples a DNA polymerase to a variable resistance component and also forms part of the variable resistance component. The DNA polymer tether 2402 includes, in certain embodiments, a linker 2404 through which the DNA polymer tether couples to the DNA polymerase, a pre-reporter region 2406 that includes a sufficient number of deoxynucleotide monomers that span a distance from the attachment point on the polymerase fragment and the reporter region when the polymerase fragment seats in the porin, a reporter region 2408 that typically includes at least four deoxynucleotide monomers, and a post-reporter region that may include a few to tens of deoxynucleotide monomers 2410. The reporter region 2408 is a region that is within a constriction in the porin channel when the DNA polymerase exhibits a variety of different conformations, exhibits different positions relative to the porin, and / or exhibits different orientations relative to the porin that together include a mechanical change that is indicated by the mechanical change sensor component of the second sequence detection system. The lengths of the various DNA polymer tether regions may vary with embodiments that use different porins and / or different DNA polymerases. The region is defined by the distance between the attachment point of the DNA polymer tether to the DNA polymerase and the narrow constriction in the porin channel, as well as the displacement of the position of the DNA polymer tether induced by the conformational change of the DNA polymerase.

[0079] As shown in the bottom of FIG. 24, the DNA polymer tether has a certain amount of dimensional flexibility, similar to an elastic or spring member in a mechanical system. When a relatively low voltage is applied to the cell 2412, the portion of the DNA polymer tether within the porin channel constriction when the DNA polymer tether is in an extended or contracted state 2418 may be different than the portion of the tether within the porin channel constriction when a higher voltage is applied to the cell 2416. A given deoxynucleotide monomer 2420 may be present at the boundary between the pre-reporter region and the reporter region in the relaxed state, whereas the same deoxynucleotide monomer may be fully present within the reporter region in the extended or contracted state. By varying the applied voltage to the cell, relatively fine adjustments can be made to the rest or baseline position of the DNA tether, and in particular the rest or baseline position of the reporter region relative to the narrow constriction of the porin channel. This provides a mechanism for calibrating the cell with respect to the magnitude of the output voltage and the mechanical to electrical transduction responsiveness of the cell.

[0080] Different sequences of deoxynucleotide monomers in the reporter region confer different resistances to ion-current flow through the porin channel. Experiments with DNA-polymer tethers with different reporter region deoxynucleotide monomer sequences have led to the identification of a number of low current, high resistance reporter region sequences and a number of high current, low resistance reporter region sequences. Figure 25 shows a number of examples of low current, high resistance and high current, low resistance four deoxynucleotide monomer sequences. Low current sequences are shown in the first column 2502 and high current sequences are shown in the second column 2504. "X" represents a baseless, broken deoxynucleotide 2506. Thus, by varying the deoxynucleotide-monomer sequence of the reporter region, one can precisely design the resistance profile of a variable resistance component for a DNA-polymer tether in a porin channel. It should be noted that high resistance deoxynucleotide-monomer sequences exhibit a resistance to current flow through the porin channel that is greater than the average resistance exhibited by a variety of different possible deoxynucleotide-monomer sequences, while low resistance deoxynucleotide-monomer sequences exhibit a resistance to current flow through the porin channel that is less than the average resistance exhibited by a variety of different possible deoxynucleotide-monomer sequences.

[0081] Figures 26A-26B show, in one example, tuning the responsiveness of mechanical-to-electrical signal transduction via DNA polymer tether sequence design. In both Figures 26A and 26B, reporter regions 2602 and 2632 are shown on the left hand side of the diagram, and the corresponding output signals are plotted in the right hand portion of diagrams 2604 and 2605. Pairs of arrows, such as pair of arrows 2606-2607, indicate the position of a narrow constriction in the porin channel relative to the reporter region in a reference position. In this example, the reporter region can move up and down in the porin channel at various distances or displacements, as indicated by double-headed arrow 2608. Current signal plot 2604 has a horizontal axis 2610 representing the displacement of the reporter region relative to the reference position, and a vertical axis 2612 representing the magnitude of current flow through the porin channel. Reporter region 2602 is represented by a series of labeled squares, such as square 2614. Each square represents a single deoxynucleotide monomer or a short sequence of deoxynucleotide monomers. The letter "L" represents low current, the letter "M" represents medium current, and the letter "H" represents high current. The reporter region 2602 in FIG. 26A changes resistance slowly and symmetrically from a low current high resistance section 2616 to a high current low resistance section 2618 to a low current high resistance section 2620. As a result, the current signal increases relatively slowly from a small negative, or upward, displacement 2620 to a larger positive or downward displacement 2624. During operation of the cell, the reporter displacement increases d min 2626 to d max2628, the magnitude of the current flow falls within the response range indicated by the double-headed arrow 2630. In contrast, reporter region 2632 in FIG. 26B is characterized by abrupt drops and rises in resistance, thus generating a very steep current increase 2634 and a corresponding larger current response range 2640 at similar displacement ranges 2636 and 2638. Thus, the current flow response to reporter region displacements due to conformational changes in the DNA polymerase can be precisely tailored through careful design of the deoxynucleotides within the DNA polymer tethered reporter region. In general, steeper and more nonlinear response curves enhance sensitivity to conformational changes in the DNA polymerase, allowing detection of dynamic oscillations and relative interdomain motions by the DNA polymerase that may be further modified by interactions between the DNA polymerase and the MspA porin.

[0082] FIG. 27 shows, in one example, a DNA polymer tether featuring a repeating deoxynucleotide sequence. FIG. 27 uses the same diagrammatic conventions used in FIGS. 26A-26B. DNA-polymer-tether sequence 2702 includes a repeating sequence of low, medium, and high current deoxynucleotide tether moieties. Arrows 2704-2706 indicate the boundaries between the repeating sequences. Arrow 2708 represents the reference position of the DNA polymer tether relative to the porin channel constriction. A plot 2710 of the current signal generated by displacing the DNA polymer tether up and down from the reference displacement 2708 shows the corresponding repeating signal morphology 2712. An advantage of the repeating resistor sequence tether is that, as discussed above with reference to FIG. 24, by adjusting the voltage applied to the cell, a current flow corresponding to a desired position on the output signal curve can be obtained regardless of the rough position of the DNA polymer tether within the porin channel. In other words, tuning the applied voltage can be viewed as a fine-tuning of the cell response, and due to the nature of the repeating sequence of the DNA polymer tether, the fine-tuning is sufficient to select one of many identical optimal baseline positions of the DNA polymer tether, even when it is difficult or impossible to determine the absolute position of the DNA polymer tether within the porin channel.

[0083] As discussed above with reference to Figures 6A-10, various derived values ​​are generated by the analysis subsystem of the sequence detection system for use in identifying objects or entities within the target. For certain embodiments of the second sequence detection system, the average current magnitude and standard deviation within the central portion of the voltage signal generated by the current-to-voltage converter circuit, discussed above with reference to Figures 14A-14B, are generated by the analysis subsystem of the voltage-signal curve generated for each deoxynucleotide added to the copy strand. However, further techniques are required to identify each added deoxynucleotide.

[0084] 28A-28C show, in one example, an approach used to distinguish different deoxynucleotides added to the copy strand by a DNA polymerase in certain embodiments of a second sequence detection system. FIG. 28A shows a plot of the mean-voltage-magnitude range of the four types of deoxynucleotide triphosphates within the active side of the DNA polymerase. All four ranges overlap to some extent, and the average voltage-signal-magnitude ranges of deoxythymidine triphosphate, deoxyadenosine triphosphate, and deoxycytosine triphosphate overlap significantly with each other. FIG. 28B shows a two-dimensional plot using both the mean voltage and standard deviation, as in FIG. 9 discussed above. The two-dimensional plot effectively distinguishes deoxyguanosine triphosphate 2802 from deoxycytosine triphosphate 2804, while the area ranges of deoxythymidine triphosphate and deoxyadenosine triphosphate are essentially coextensive 2806. In order to fully distinguish between the four deoxynucleotide triphosphates, a modified adenine base 2810, 7-deazaadenine, is used in place of adenine 2812. When the modified deoxyadenosine-like triphosphate is present within the active site of a DNA polymerase, the DNA polymerase occupies a conformation that is significantly different than the conformation that the DNA polymerase occupies when deoxyadenosine triphosphate is present within the active site. As a result, the area extent of the modified deoxyadenosine-like triphosphate 2814 in the two-dimensional plot 2816 is no longer coextensive with the area extent of the deoxythymidine triphosphate 2818, and all four different deoxynucleotide triphosphates can be clearly identified based on the mean voltage signal and standard deviation derivatives that it generates from the output voltage signal it generates when present in the active site.

[0085] Figures 29A-29C show, in one example, the use of a locking oligonucleotide as a locking component to hold a DNA polymer tether tightly within a porin channel. Figure 29A shows a DNA polymer tether with a locking oligonucleotide, using the same diagrammatic conventions previously used in Figure 24. As shown in Figure 29A, the locking oligonucleotide 2902 has a deoxynucleotide-monomer sequence complementary to a portion of the deoxynucleotide monomer sequence of the DNA polymer tether 2904, and hybridizes to a portion of the DNA polymer tether via hydrogen bonding and base stacking. Figures 29B-29C show the use of a locking oligonucleotide during cell operation. Initially, the DNA polymerase 2906 and DNA polymer tether 2908 are not associated with the porin 2910. Applying a voltage across the cell 2912 threads the DNA polymer tether through the porin channel 2914, sedate the DNA polymerase through it and into the pore 2916. The locking oligonucleotide then associates with the portion of the DNA-polymer tether 2918 that extends into the second solution-containing chamber of the cell. As shown in FIG. 29C, the locking oligonucleotide prevents the DNA polymer tether from being pulled out of the porin channel 2920 when a voltage of reverse polarity 2922 is applied to the cell. As a result, restoring the original polarity of the applied voltage 2924 re-seats the DNA polymerase within the porin 2926. However, application of a much larger voltage 2928 generates sufficient force to strip the locking oligonucleotide 2930 from the DNA polymer tether 2932 and completely dissociate the DNA polymerase and DNA polymer tether from the port. In certain embodiments, locking components other than locking oligonucleotides may be used. For example, a biotin moiety may be attached to the tether and streptavidin may be used as the locking component. Other types of biopolymer locking components may also be used in alternative embodiments.

[0086] The use of locking oligonucleotides to tether the DNA polymer within the porin channel provides several important advantages. For certain types of DNA polymerases, the voltage signal generated by the DNA polymerase and the DNA polymer tether-based variable resistance component will experience extended periods of increased noise, masking fluctuations in the current signal used to distinguish between different types of deoxynucleotide triphosphates that specifically bind to the active site of the DNA polymerase. To prevent this type of noise generation, in certain embodiments, the polarity of the applied voltage is periodically reversed to disengage the DNA polymerase from the porin. The polarity of the applied voltage is then reversed again to begin the next interval of sequence detection. FIG. 30 illustrates an applied voltage cycle that, in one embodiment, is used to prevent the generation of noise that disrupts the output voltage signal. In the plot shown in FIG. 30, the horizontal axis 3002 represents time and the vertical axis 3004 represents the applied voltage. The applied voltage alternates between relatively long periods of relatively large applied voltage of a normal first polarity, such as interval 3006, and relatively short periods during which the polarity of the applied voltage is reversed, such as interval 3008, as shown in FIG.

[0087] Although the sequence detector and other systems disclosed herein are described in terms of specific embodiments, it is not intended that the invention be limited to these implementations. Modifications within the spirit of the invention will be apparent to those skilled in the art. For example, certain sequence detection systems can use different types of mechanically altered sensor components, mechanical couplers, variable resistance components, and electrical signal generating components and circuits. For example, the second sequence detection system described above can use different DNA polymerases and DNA polymerase fragments, use different porins, use different types of mechanical couplers that include different types of polymers, and use cells of many different shapes, sizes, and configurations. Examples of polypeptide pores that can be used in place of MspA-porin include α-hemolysin, Mycobacterium smegmatis porin A, gramicidin A, maltoporin, OmpF, OmpC, PhoE, Tsx, F-pilus, SP1, mitochondrial porin (VDAC), Tom40, outer membrane phospholipase A, and Neisseria autotransporter lipoprotein (NaIP).Examples of polymerases or fragments thereof that can be used in place of the Klenow fragment discussed above include T7 DNA polymerase, eukaryotic mitochondrial DNA polymerase gamma, E. coli DNA Pol I, Thermus aquaticus Pol I, Bacillus stearothermophilus Pol I, eukaryotic DNA polymerases alpha, delta, and epsilon, DNA polymerase zeta, T4 DNA polymerase, Phi29 DNA polymerase, RB69 bacteriophage DNA polymerase, E. coli DNA polymerase III alpha subunit, polymerases from the archaeal Euryarchaeota subdomain, eukaryotic polymerases Pol beta, Pol sigma, and Pol μ, S. cerevisiae Pol 4, Pol eta, Pol iota, Pol kappa, E. coli Pol IV (DINB), E. coli Pol V (UmuD'2C), retroviral reverse transcriptase and eukaryotic telomerase, T7 Included are viral RNA polymerases such as RNA polymerase, eukaryotic RNA polymerases such as RNA polymerase I, RNA polymerase II, RNA polymerase III, RNA polymerase IV, RNA polymerase V, and archaeal RNA polymerase. Alternative examples of tethers include synthetic polymers such as PEG (polyethylene glycol), PPG (polypropylene glycol), PVA (polyvinyl alcohol), PE (polyethylene). Additionally, DNA, RNA, PNA or LNA labels, morpholino, or chiral DNA, and the like. Additionally, in exemplary embodiments, iso-G and iso-C bases may be present on the tether. A variety of different values ​​derived from the output voltage signal can be used as different dimensions to uniquely identify the type of object or entity within the target sequence. The magnitude of the average voltage signal, the variance calculated from the signal voltage, and the standard deviation are just a few examples of the various types of derived values ​​that can be used to identify the type of object or entity. In addition to 7-deazadeoxyadenosine triphosphate, many different types of modified deoxynucleotide triphosphates can be used for copy strand extension.Because copy strand extension is used solely to determine the template strand sequence, the chemical composition of the copy strand produced during sequencing is irrelevant to the correct operation of the second sequence detection system. All combinations of the concepts, components, and methods discussed are contemplated and understood.

[0088] It is to be understood that all combinations of the foregoing and additional concepts (unless such concepts are mutually inconsistent) are contemplated as part of the inventive subject matter disclosed herein. In particular, all combinations of claimed subject matter appearing at the end of this disclosure are considered to be part of the inventive subject matter disclosed herein.

Claims

1. a mechanical change sensor that exhibits one or more mechanical changes when specifically interacting with an entity within the target, where each entity has a type; A mechanical change-to-signal transducer that converts one or more mechanical changes into a signal; and An analysis subsystem that uses the signal to determine the type of entity within the target. A detection system comprising:

2. The analysis subsystem consists of: For each portion of the electrical signal that corresponds to a particular entity within the target, calculating n derivatives from a portion of the electrical signal, where n is an integer greater than or equal to 2; Mapping two or more derived values ​​to an n-dimensional range volume that corresponds to a particular entity type; and Assigning an entity to a specific entity type 10. The sequence detection system of claim 1, wherein the electrical signal is used to determine the type of entity in the target by detecting the electrical potential of the entity.

3. The n derivative values ​​are as follows: Average of multiple signal magnitude samples; the variance of a plurality of signal magnitude samples; and Standard deviation of multiple signal magnitude samples The sequence detection system of claim 2 , comprising one or more of the following:

4. The sequence detection system of claim 2, wherein the analysis subsystem stores a representation of the sequence of entity types assigned to corresponding sequences of entities in the target in the memory for at least one of subsequent retrieval, transmission, display, and further analysis.

5. Mechanical change – signal transducers are A variable resistor to regulate the flow of current; a mechanical coupler coupling the mechanically changeable component to the variable resistor; and A current-to-voltage converter that outputs a voltage signal that varies according to the rate of current flow. The sequence detection system of claim 1 , further comprising:

6. The target is a primer-associated DNA template polymer; The mechanically altering component is a DNA polymerase; The mechanical coupler is a DNA polymer tether with one end attached to a DNA polymerase; and The sequence detection system of claim 5 , wherein the variable resistor is a reporter region of a DNA polymer tether located within a constriction in the porin channel.

7. A mechanistically altering component of a nucleic acid polymerase that exhibits a mechanistic change when it specifically associates with a nucleotide polyphosphate in the active site; A mechanical change-signal transducer that converts the mechanical change of the mechanical change component of the nucleic acid polymerase into an output signal; and An analytical subsystem that uses the output signals to determine the sequence of monomer types within the nucleic acid polymer target. A sequence detection system comprising:

8. Mechanical change – signal transducers: a tether having one end that binds to a mechanically modifiable component of a nucleic acid polymerase and that includes a reporter region that translocates through a constriction of the porin channel when the mechanically modulated component of the nucleic acid polymerase undergoes a mechanical change, thereby variably modulating the flow of ions through the porin channel that constitutes an electric current; and A current-to-voltage converter that outputs a voltage signal that varies in response to the rate of current flow through the porin channel. The sequence detection system of claim 7 , further comprising:

9. 9. The sequence detection system of claim 8, wherein the reporter region is an oligonucleotide, and different nucleotide subsequences within the reporter region provide different resistances to current flow when positioned within the constriction of the porin channel.

10. 10. The sequence detection system of claim 9, wherein the reporter region is an oligodeoxynucleotide, and the reporter region exhibits a resistance to current flow that is greater than the average resistance for oligodeoxynucleotides of similar length.

11. 10. The sequence detection system of claim 9, wherein the reporter region is an oligodeoxynucleotide, and the reporter region exhibits a resistance to current flow that is lower than the average resistance for oligodeoxynucleotides of similar length.

12. the sensitivity of the output signal to conformational changes of the mechanically altered component of the nucleic acid polymerase increases with increasing spatial rate of change of resistance to current flow provided by the reporter region; and 10. The array detection system of claim 9, wherein the reported area has a spatial variation in resistance that produces a particular output voltage signal response.

13. the tether exhibits dimensional flexibility; and 10. The sequence detection system of claim 9, wherein modulation of the voltage applied across the porin channel translocates the reporter region within the porin channel.

14. the resistance of the reporter region to current flow is spatially periodic; and 14. The sequence detection system of claim 13, wherein the sequence detection system is calibrated to have a particular output voltage signal response by adjusting the voltage applied across the porin channel.

15. The sequence detection system of claim 13, wherein the locking component associates with a portion of the polymer tether that extends beyond the porin channel on the opposite side of the porin channel from the mechanical change component of the DNA polymerase to secure the polymer tether within the porin channel.

16. 16. The sequence detection system of claim 15, wherein the polarity of the voltage applied across the porin channel is periodically reversed to reduce noise in the output signal.

17. a first chamber containing a buffered solution of ionizable salts, deoxynucleotide polyphosphates, and primer-associated DNA template polymers; a second chamber containing a buffer solution of an ionizable salt; and A barrier isolating a first chamber from a second chamber, the barrier having a porin channel providing fluid communication between the first chamber and the second chamber. a two-chamber cell comprising: a first electrode in fluid contact with the buffer solution in the first chamber and connected to a current-to-voltage converter; and a second electrode in fluid contact with the buffer solution in the second chamber and connected to a current-to-voltage converter; The sequence detection system of claim 7 , further comprising:

18. 1. A method for determining a monomer sequence from a signal output by a sequence detection system, comprising: identifying portions of the signals each corresponding to a different monomer in the sequence of monomers; For each signal part, calculating n derivatives from the signal portion, where n is an integer greater than or equal to 2; Mapping the n-derived values ​​to a volume of n-dimensional range corresponding to a particular type of monomer; and Assigning a particular type of monomer to a signal portion; and Generate and store a symbolic representation of the sequence of monomer types complementary to the monomer types assigned to the signal moieties. The method includes:

19. The n derivative is: Average of multiple signal magnitude samples; the variance of a plurality of signal magnitude samples; and Standard deviation of multiple signal magnitude samples 20. The method of claim 18, comprising one or more of the following:

20. 20. The method of claim 18, wherein the sequence of monomers is a sequence of deoxynucleotide monomers, one or more of which are chemically distinct from deoxyadenosine, deoxyguanosine, deoxythymidine and deoxycytidine.

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