Adjustment and calibration features of the sequence detection system

Abstract

To provide new and different types of sequence detectors to facilitate identifying and deploying specific sequence-detection processes and systems that best meet sequence-detection parameters and goals for specific applications.SOLUTION: The current specification discusses electromechanical sequence detectors that transduce changes in the shape of a shape-change sensor component into an electrical signal from which one or more derived values are generated. In a disclosed implementation, a sequence-detection system comprises: a mechanical-change sensor that changes the shape when specifically interacting with entities within a target; a shape-to-signal-transduction component that transduces changes in the shape of the mechanical-change sensor into an electrical signal; an analysis subsystem that determines types of entities within the target using the electrical signal; and a control subsystem that continuously monitors operational characteristics of the sequence-detection system and adjusts operational parameters of the sequence-detection system.SELECTED DRAWING: Figure 13

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 from the detectors to identify the types of entities and objects 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, volumetric 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. In many cases, 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 specific applications. 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-signal transducer 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-signal transducer 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 monomer types in a nucleic acid polymer target. The specification further discusses a method for determining a monomer sequence from a signal output by a 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 ​​from the signal portions, where n is an integer equal to or greater than 1, 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 monomer types complementary to the monomer types assigned to the signal portions. In certain cases, the signal derived values ​​may be sufficient to identify the different monomers in the 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 representation 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] [Diagram 3] 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 array detector 204 shown in FIG.

[0009] [Figure 6] 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 shows, in one example, a mechanical variation-based sequence detector included in the second sequence detection system.

[0017] [Figure 14A-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.

[0018] [Figure 15] FIG. 15 shows, in one example, an array of cells that provides parallel sequencing.

[0019] [Figure 16] FIG. 16 shows a short DNA polymer.

[0020] [Figure 17A-17B] 17A-17B show hydrogen bonds between purine and pyrimidine bases of two opposing parallel DNA strands.

[0021] [Figure 18] 18A-18B show double-stranded DNA.

[0022] [Figure 19] Figure 19 shows the names and chemical structures of the 20 common amino acids.

[0023] [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.

[0024] [Figures 21A-21E] 21A-21E show copy strand elongation catalyzed by the Klenow fragment of E. coli DNA polymerase I.

[0025] [Fig. 22A-22B] 22A-22B show, in one example, variable resistance components of a second array detection system.

[0026] [Diagram 23] FIG. 23 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.

[0027] [Figure 24] FIG. 24 illustrates, in one example, the adjustment of a cell's signal response.

[0028] [Diagram 25] FIG. 25 shows, in one example, the generation of periodic noise in a second type of sequence detection system when the Klenow fragment of E. coli DNA polymerase I is used as the shape-change sensor component.

[0029] [Figure 26] Figure 26 shows copy strand elongation catalyzed by the Klenow fragment of E. coli DNA polymerase I.

[0030] [Figure 27] FIG. 27 shows, in one example, a primer-associated template DNA strand configured for determination of the deoxynucleotide-monomer type at a single position.

[0031] [Figure 28] FIG. 28 lists, in one example, various types of parameters that may be adjusted to configure, calibrate and tune the operation of a second type of sequence detection system.

[0032] [Figures 29A-29C] Figures 29A-29C show, in one example, the use of locking oligonucleotides to securely hold a DNA polymer tether within a porin channel.

[0033] [Fig. 30A-30D] 30A-30D provide a control flow diagram illustrating an example of the control of one embodiment of the second type of sequence detection system. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0034] Discussed herein are array detectors that generate signals that can determine the sequence of entity types in a target. Array detectors described herein can 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 array detector detects the sequence by generating signals that can determine the sequence of the entity types in the target. The array detector may include components, such as a microprocessor-controlled signal analysis component, that analyzes the signal to determine the sequence of the entity types. The target includes an array of entities, each entity having a type. The array detector physically interacts with the target and generates signals that change when a mechanical change sensor in the array detector specifically interacts with a different type of entity in the target. A specific interaction is an interaction between a 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-signal transducer into a corresponding signal, and the corresponding signal is also characteristic of the entity type to which the entity belongs. Mechanical changes may include mechanical changes--changes in shape and / or size of the signal transducer, mechanical changes--changes in position of the signal transducer relative to other components, mechanical changes--changes in orientation of the signal transducer 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 strings or wires. 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 when the target passes through the mechanical change sensor, and the mechanical change sensor specifically interacts with each object, causing a mechanical change in the mechanical change sensor that is converted into an electrical signal characteristic of the shape of the object by the variable resistor. The second example target is a biopolymer that includes an array of monomers that are covalently bonded 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, and additional types of monomers occur less frequently. The second corresponding array detector generates a time-varying electrical signal when the target biopolymer passes through the mechanical change sensor, and the mechanical change sensor specifically associates with the monomers, causing a mechanical change in the mechanical change sensor, which is then communicated to the variable resistor by the coupler. These two types of detectors are examples, and other configurations exist and can be implemented. First Type of Mechanical Change-Based Electromechanical Array Detector

[0035] FIG. 1 illustrates, in one example, a target input to a first (type) mechanical variation based array detector, and a determined array 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 array detector, which outputs a symbolic representation of the array of object types 132 within the target.

[0036] 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 computer system 212, processes the electrical signal to determine the 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.

[0037] 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.

[0038] 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.

[0039] Figure 5 illustrates, in one example, the internal components of the mechanical change based array detector 204 shown in Figure 2. The mechanical change based array detector includes the mechanical change-detection subsystem 402 shown in Figure 4 and a pair of counter-rotating gear drums 502 and 504 driven by two electric motors that feed a target 506 object through the mechanical change sensor component 302. The pair of counter-rotating gear drums feed 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).

[0040] 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.

[0041] 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:

[0042]

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[0043]

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[0044]

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[0045]

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[0046] 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.

[0047] 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.

[0048] In some instances, if a particular output signal is insufficient to distinguish between different types of objects in an array, the array detector designer may seek 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 may use 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.

[0049] 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.

[0050] 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

[0051] 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.

[0052] 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.

[0053] FIG. 13 illustrates, in one example, a mechanical change-based array detector included in a second array detection system. In the following discussion, the mechanical change-based array 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 array detector, rather than accurately depicting the relative scale of the components.

[0054] 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.

[0055] 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:

[0056] 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.

[0057] 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.

[0058] 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.

[0059] 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.

[0060] 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.

[0061] 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.

[0062] 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.

[0063] 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.

[0064] 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.

[0065] 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.

[0066] 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.

[0067] 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 of the 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 the DNA-polymerase is dynamic and is generally associated with various types of subtle vibrational modes and relative movements 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 position or orientation of the DNA-polymerase and the porin, and / or other changes that are believed to be the source of the mechanical signal of the mechanical changes generated by the DNA polymerase acting as a mechanical change sensor component of the second sequence detection system.

[0068] 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.

[0069] 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.

[0070] 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. Calibration and tuning of a second type of shape-change based array detector

[0071] FIG. 23 illustrates some features of a 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. The DNA-polymer tether 2302 includes a linker 2304 where the DNA polymer tether attaches to the DNA polymerase, in certain embodiments, a pre-reporter region 2306 that includes about six deoxynucleotide monomers, a reporter region 2308 that typically includes at least four deoxynucleotide monomers, and a post-reporter region that includes several to several tens of deoxynucleotide monomers 2310. The reporter region 2308 is a region that is within a narrow constriction in the porin channel where the DNA polymerase exhibits a variety of different conformations that together include a shape change exhibited by a shape change sensor component of a second sequence detection system. The lengths of the various DNA polymer tether regions may vary with different 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 of the porin channel, and the range of displacement of the position of the DNA polymer tether induced by the conformational change of the DNA polymerase.

[0072] As shown in the bottom of FIG. 23, DNA polymer tethers generally have some degree of dimensional flexibility. When a relatively low voltage is applied to cell 2312, the DNA polymer tether settles into one of a variety of relaxed states 2314, whereas when a high voltage is applied to cell 2316, the DNA polymer tether settles into one of a variety of stretched or tensioned states 2318. In the relaxed state, a given deoxynucleotide monomer 2320 may reside at the boundary between the pre-reporter and the reporter region, whereas in the stretched or tensioned state, the same deoxynucleotide monomer may reside in a well within the reporter region. By varying the voltage applied 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 constriction of the porin channel. This provides a means of calibrating the cell with respect to the magnitude of the cell's output voltage and the responsiveness of mechanical-to-electrical signal transduction.

[0073] Different sequences of deoxynucleotide monomers in the reporter region provide different resistances to ion-current through the porin channel. Various experiments with DNA polymer tethers with different reporter region deoxynucleotide monomer sequences lead 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. The variable resistance profile of the variable resistance component of the machine translation of DNA-polymer tethers in porin channels can be precisely engineered by varying the deoxynucleotide-monomer sequences of the reporter region.

[0074] FIG. 24 illustrates, in one example, the tuning of the signal response of a cell. The DNA-polymerase mechanical change component may occupy different positions relative to the porin channel and may adopt different conformations under different conditions and cell parameter values, resulting in different positions of the reporter region relative to the porin contraction. A hypothetical reporter region 2404 is shown on the left hand side of FIG. 24. As discussed above, the reporter region moves up and down within the porin channel. Movement is characterized as negative displacement, or upward displacement, and positive displacement, or downward displacement, from a reference displacement 2404, considered to have a value of 0, d0. A plot 2406 of signal intensity with respect to the displacement of the reporter, or voltage magnitude of the output signal, is shown on the right hand side of FIG. 24. The vertical axis 2408 represents the percentage of current flowing through the porin channel, or the magnitude of the output voltage signal, and the horizontal axis 2410 represents the displacement of the reporter within the porin channel relative to the reference displacement d0. Reporter region 2402 is represented by a series of labeled squares, such as square 2412. 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. Current signal curve 2414 mirrors the current representation of the reporter region 2402 representation and inversely mirrors the resistivity of the deoxynucleotide monomers, or short sequence of deoxynucleotide monomers, represented by small squares in the reporter region 2402 representation. In other words, if the reporter region representation were rotated 90° counterclockwise and overlapped with the horizontal axis 2410 of plot 2406, the current representation of each deoxynucleotide monomer or sequence of deoxynucleotide monomers and their location along the displacement axis can be used as vertical and horizontal coordinates to place points on the plot and, when connected with a continuous curve, would produce the current signal curve 2414 shown in plot 2406. The steepest portion 2420 of the current signal curve 2414 occurs when the displacement coordinate is d, as shown by the vertical dashed line 2422 in FIG. 0にThe reporter is bisected at an equal point. Generally, this reference position represents the desired resting or baseline point for the reporter in the porin channel because it provides the greatest sensitivity in voltage signal output by the cell to conformational changes in the DNA-polymerase mechanical change sensor component. Because the reporter and porin channel are nanoscale components, it is difficult to mechanically configure the cell to position the reporter at a desired reference displacement position in the porin channel. However, as discussed above with reference to FIG. 23, varying the voltage applied to the cell can finely tune the baseline or resting position of the reporter in the porin channel. Because the shape and morphology of the current-signal curve 2414 are determined by the sequence of deoxynucleotide monomers in the reporter region, the response of the cell can be initially calibrated by positioning the reporter region at the desired reference displacement and varying the voltage applied to the cell to bisect the steepest portion of the current-signal curve. Thus, the current response to reporter region displacement due to conformational changes in DNA polymerase can be precisely tuned by careful design of the deoxynucleotide sequences within the DNA polymer-tethered reporter region, and the current response is calibrated by varying the voltage applied to the cell.

[0075] FIG. 25 illustrates, in one example, the occurrence of periodic noise in a second type of sequence detection system when the Klenow fragment of E. coli DNA polymerase I is used as the mechanical change sensor component. This type of noise has been observed when another DNA-polymerase mechanical change sensor is used in the second type of sequence detection system. FIG. 25 illustrates a plot 2502 of a current signal, represented by the vertical axis 2504, with respect to time, represented by the horizontal axis 2506. During certain periods 2508-2510, the average current flow is at a relatively low value 2512. However, during other periods 2514 and 2515, the value of the average current flow is at a significantly higher value 2518. In addition, the range of deviations to the average current flow during the low current flow periods 2508-2510 is much smaller than the range of deviations to the average current flow during the high current flow periods 2514 and 2515. The current signal curve containing the data is superimposed on the noisy signal 2520 shown in FIG. 25. Current signal curves containing data are easily extracted from the current signal output during the low noise, low current flow periods 2508-2510, but not from the noisy current signal output during the high current flow periods 2514 and 2515. This is because noise inherently masks relatively small information-carrying current fluctuations. The low noise, low current periods 2508-2510 are labeled "measurement" in FIG. 25, while the high current flow periods 2514 and 2515 are labeled "noise". If steps are not taken to reduce or eliminate the noisy periods, a large amount of sequence information is lost. This is because copy strand extension by the DNA-polymerase mechanical change sensor component continues during the noisy periods in which the output voltage of the mechanical change sensor component to conformational changes in DNA-polymerase cannot be extracted from the output voltage signal. Even ignoring the loss of sequence information, periods of sequence information masking noise represent a significant reduction in the rate of sequence information acquisition.However, as discussed further below, the term sequence information masking noise has the advantage that sequence information masking noise only occurs when the DNA polymer tether is threaded through the porin channel and DNA polymerase is associated with the porin, thus indicating that the mechanical change sensor component and the variable resistance component are first configured.

[0076] Figure 26 shows copy strand elongation catalyzed by the Klenow fragment of E. coli DNA polymerase I. Active sites within the DNA polymerase are represented by vertical rectangles 2602-2607 at various times during the incorporation of nucleotide triphosphates into the copy DNA strand. The time points represented by these active site representations are shown along a timeline 2610. One complete cycle of nucleotide triphosphate incorporation spans the time segment 2612 shown below the timeline. Figure 26 uses the same diagrammatic conventions used in Figures 21A-E. Hydrogen bonds are represented as in Figures 21A-E, but because the identity of the base pair nucleotides is not specified, three short horizontal line segments generally represent hydrogen bonds, whether two hydrogen bonds in an AT base pair or three hydrogen bonds between a CG base pair. Portions of the template DNA strand 2616 and copy DNA strand 2618 are represented by rectangles and disks. Purine and pyrimidine bases are represented by long, vertical rectangles, such as rectangle 2620. The ribose moiety in each deoxynucleotide monomer is represented by a small square, such as small square 2622. The phosphodiester bonds that join two deoxynucleotide monomers in a chain are represented by a circle, such as circle 2624.

[0077] Initially, the active site 2602 is ready to receive the next deoxynucleotide triphosphate for addition to the 3' end of the copy DNA strand 2618. Next, the deoxynucleotide triphosphate 2626 approaches the active site. Of course, any of the four different deoxynucleotide triphosphates may approach the active site, but the affinity of the deoxynucleotide triphosphate for the active site that is complementary to the unpaired deoxynucleotide monomer of the template strand within the active site is generally higher than the binding affinity of the non-complementary deoxynucleotide triphosphate, and the binding affinity of the complementary deoxynucleotide triphosphate is most likely to specifically associate with the unpaired active site resident template strand deoxynucleotide folder by hydrogen bonding the active site 2628. This time required for the specific association of the next nucleotide triphosphate with the active site is influenced by many cell parameters, including the concentration and type of nucleotide triphosphate in the cell chamber solution, and the temperature. Next, the polymerization reaction 2630-2631 shown in Figure 20 occurs, forming a phosphodiester bond bridge 2632 that incorporates the new deoxynucleotide monomer into the copy strand. The time required for this reaction is affected by many cell parameters including, for example, the type of nucleotide triphosphate in the cell chamber solution, temperature, the concentration of magnesium ions in the cell chamber solution, the pH of the cell chamber solution, and the concentrations of other ionized salts in the cell chamber solution. Inorganic pyrophosphate 2634 is released from the active site. DNA polymerase translates against the template, copying the DNA strand to reform the active site 2607 free of nucleotide triphosphate and ready for the specific association of subsequent deoxynucleotide triphosphates. This translation is affected by various cell parameters such as temperature, in one example.

[0078] The length of the nucleotide triphosphate-addition cycle, represented by line segment 2612, is an important parameter with respect to the operation of the second type of sequence detection system. In order to extract reliable data from the output voltage signal by the analysis subsystem, the conformational change of the DNA-polymerase mechanical change sensor component must occur for a sufficient period of time to allow the current-to-voltage converter data sampling circuit to detect the conformational change in the corresponding current flow change and generate a current signal containing sufficient information to allow the type of nucleotide triphosphate to be determined by signal processing in the analysis subsystem. However, the rate of sequencing is directly proportional to the rate at which nucleotide triphosphates are incorporated into the copied DNA strand. As discussed below, there are many different adjustable parameters associated with the second type of sequence detection system that can be adjusted to provide a desired nucleotide triphosphate addition cycle length that is long enough to ensure accurate DNA template sequencing, but short enough to ensure that the desired rate of sequencing is achieved.

[0079] In certain embodiments, it may be desirable to configure a second type of sequence detection system to determine only the type of deoxynucleotide monomer at a particular single position within the DNA template. FIG. 27 shows, in one example, a primer-associated template DNA strand configured for the determination of the deoxynucleotide-monomer type at a single position. In FIG. 27, the same diagrammatic conventions used in FIG. 26 are used to represent the primer DNA 2702 and the primer-associated template DNA strand 2704. For certain DNA polymerases, an RNA primer may be used instead of a DNA primer. In either case, the primer may include a dideoxynucleotide monomer 2710 at the 3′ end. Because the dideoxynucleotide monomer lacks a 3′ hydroxyl, it cannot be extended by the addition of a nucleotide triphosphate via the nucleotide triphosphate addition reaction discussed above with reference to FIG. 20. However, as shown in the active site representation 2604 of FIG. 26, a complementary nucleotide triphosphate may nevertheless associate with the first unpaired deoxynucleotide monomer 2712 of the primer-associated template DNA strand. It is the specific association of a complementary nucleotide triphosphate at the active site that induces a conformational change in the shape-change sensor component of the DNA polymerase that generates a signal. Thus, a primer-associated template DNA strand having a dideoxynucleotide monomer at the 3' end of the primer will generate only a single voltage-signal curve that is indicative of the type of nucleotide triphosphate that is complementary to the first unpaired deoxynucleotide monomer 2712 of the primer-associated DNA strand. In another embodiment, a deoxynucleotide or ribonucleotide moiety is present at the 3' end of the primer. To prevent incorporation of the nucleotide triphosphate, e.g., Mg 2+ Or Mn 2+ There are no catalytic divalent metal cations such as Ca at concentrations between 0.5 mM and 10 mM. 2+ Or Sr 2+ Use non-catalytic divalent metals such as

[0080] FIG. 28 lists, in one example, different types of parameters that may be adjusted to configure, calibrate, and adjust the operation of a second type of sequence detection system. The first column 2802 includes parameters with initial fixed values ​​that are generally adjustable only before the start of sequence detection, although in certain microfluidic and other embodiments, it may be possible to dynamically change the composition of the solution in the chamber containing the cell's solution, and dynamically adjust these other parameters. The second column 2804 shows parameters that have values ​​that can be adjusted during operation of the cell, but the adjustment involves a significant delay time. The third column 2806 includes continuously adjustable parameters that can be easily and directly adjusted during operation of the cell. This third group of parameters provides a basis for fine-tuning the cell by a control component during operation of the cell.

[0081] The initially fixed parameters shown in column 2802 include the type of monovalent cation, the concentration of the monovalent cation / chloride salt of one or more of the buffers used in the cell chamber, the type and concentration of the divalent cation / chloride salt used in the cell chamber, the type of pH buffer compound(s) used in the cell chamber, the type and concentration of other cell chamber buffer components including a reducing agent such as dithiothreitol, the type of DNA polymerase, the type of porin, the type and structure of the tether used to mechanically couple the DNA polymerase to and as part of the variable resistance component, the type of primer associated template, the primer associated template concentration, the type and concentration of nucleotide triphosphates, the membrane or bilayer components, the type of electrodes, the volume, shape and dimensions of the cell chamber, and various other parameters of the cell. Different types of salts can be used as electrolytes when different types of electrodes are used.

[0082] Many of the parameters initially fixed directly affect the length of the nucleotide triphosphate-addition cycle, as discussed above with reference to Figure 26. The length of the nucleotide triphosphate-addition cycle is sensitive to the type and concentration of nucleotide triphosphate, the type of polymerase, the pH of the buffer, and the concentrations of various ionized salts. In one example, in certain cases, K + Instead of Na + , Li + , Rb + , NH4 + and / or Cs + The use of cations such as 2802, 2803, 2804, 2805, 2806, 2807, 2808, 2810, 2811, 2812, 2813, 2814, 2815, 2816, 2817, 2818, 2819, 2900, 2910, 2920, 2930, 2931, 2940, 2941, 2950, ​​2951, 2960, 2970, 2971, 2980, 2982, 2990, 2991, 2992, 2993, 2994, 2995, 2996, 2997, 2998, 2999, 2999, 3000, 3001, 3002, 3003, 3004, 3005, 3006, 3007, 3008, 3009, 3001, 3009, 3001, 3001, 3002, 3003, 3004, 3005, 3006, 3007, 3008, 3009, 3001, 3009, 31001, 3101, 3102, 3103, 3104, 3105, 3106, 3107, 3108, 3109, 32001, 3201, 3201, 3201, 3201, 3201, 3201, 32

[0083] Certain parameters are adjustable during cell operation and greatly affect the length of the nucleotide triphosphate-addition cycle and many other operational characteristics of the second type of sequence detection system. These are classical parameters that are varied and controlled to obtain definitive and reproducible results of chemical reactions and systems as well as many other types of systems. These parameters include the temperature, pressure, and pH of the cell and its contents. It should be noted that the adjustable parameters associated with the lag time, as well as the initially fixed parameters, may be evaluated by experiment to select parameter values ​​that provide the desired cell operational characteristics.

[0084] The adjustable parameters include the magnitude of the voltage applied to the cell, the polarity of the applied voltage, as well as various instrument parameters related to the operation of the current-to-voltage converter circuitry and the analytical subsystem, providing the opportunity to fine-tune cell operation on microsecond, millisecond, and longer time scales. The use of these parameters to control the operation of the second type of sequence detection system is described below in a series of control flow diagrams.

[0085] Figures 29A-29C show, in one example, the use of a locking oligonucleotide to securely hold a DNA polymer tether 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 stably associates with the DNA-polymer tether via hydrogen bonding and base stacking. Figures 29B-C 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. Application of a voltage drop across the cell 2912 threads the thread of the DNA polymer tether into the porin channel 2914, and the DNA polymerase seats within 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 the opposite polarity 2922 is applied to the cell. As a result, restoring the original polarity of the applied voltage drop 2924 re-seats the DNA polymerase within the porin 2926. However, application of a voltage drop 2928 of a much larger opposite magnitude 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 group may be attached to the tether and streptavidin may be used as the locking component. In other embodiments, other types of biopolymer locking components may also be used.

[0086] 30A-30H provide a control flow diagram illustrating an example of a sequencing process using a second type of sequence detection system. The steps include automated control activities performed by control logic within the control components of the sequence detection system, including the control components implemented within the analytical subsystem, but may also include manual and semi-manual steps performed by a technician.

[0087] FIG. 30A provides a control-flow diagram for the routine "detect sequence", which represents the operation of the sequence detection system for one sequencing run in which the sequence detection system is initially configured and then operated to generate an output signal until one of several different termination conditions occurs. In step 3002, the routine "detect sequence" receives a pointer param to an array of sequence detector operational parameter values, each associated with a certain index. A particular parameter, such as a parameter including the magnitude of the voltage to be applied to the cell to generate an output signal including sequence identification information, is indicated as param[read volts], where read volts is an index for the parameter in the array of parameters indicated by the pointer param. These parameter values ​​include values ​​for the various fixed and adjustable parameters discussed above with reference to FIG. 28. The parameter values ​​also include several thresholds, discussed further below, and various timing parameters, also discussed below. In step 3004, the routine "setup cell" refers to configuring the sequence-detection cell for the sequence-identification experiment. Cell configuration may include one or more of an automatic configuration step, a semi-automatic configuration step, and a manual configuration step. The text "(param)" indicates that the pointer param is passed to the routine as an argument. In step 3006, the routine "configure sequencer" is called to prepare the sequence-detector and begin generating an output signal. The routine "configure sequencer" is discussed below with reference to FIG. 30B. If the routine "configure sequencer" returns an indication of success as determined in step 3008, then "read sequencer" is called in step 3010 to generate an output signal in which the sequence of the biopolymer introduced into the cell is determined by the analysis subsystem of the sequence detector. Otherwise, if the routine "configure sequencer" returns an indication of failure, then an error handling routine is called in step 3012.This error handling routine can report the inability to configure sequence-detectors for sequence discrimination experiments, and in certain embodiments, perform automated remediation steps to handle configuration failures and / or alert technicians and other individuals.

[0088] FIG. 30B provides a control-flow diagram of the routine "Configure Sequencer" at step 3010 of FIG. 30A. The routine "Configure Sequencer" illustrates the configuration of a sequence detection system to perform a sequencing experiment. In one embodiment, a voltage is applied to the cell and the output voltage signal is analyzed to look for periods of sequence information masking noise as discussed above with reference to FIG. 25. When sequence information masking noise is detected, the control logic infers that the DNA polymer tether has been retracted through the porin channel and that the DNA polymerase mechanical change sensor component has been anchored within the porin. At step 3016, a local variable trie is initialized to 0. At step 3018, a memory buffer represented by the array currentData is initialized with an index nxtSample indicating the next location in the memory buffer to store the signal samples. At step 3020, a voltage having a magnitude indicated by the parameter value param[Configure Voltage] and a polarity indicated by the parameter param[Configure Pol] is applied to the cell while the sampled output signal is collected in the memory buffer currentData. In step 3022, the data in memory buffer currentData is analyzed to determine whether noise above a threshold noise level indicated by the parameter value param[noiseT] is observed in the sampled signal. If such noise is observed, as determined in step 3024, the routine "configure sequencer" infers that the mechanical change-sensor component has been anchored within the porin. If the parameter value param[lock] is not equal to 0, as determined in step 3026, the tether is locked in step 3028, as discussed above with reference to Figures 29A-29C. The routine "configure sequencer" then returns with an indication of success.If no noise is observed as determined in step 3024, and if the value of local variable TRE determined in step 3030 is less than the parameter value param[retry], then a reverse polarity voltage is briefly applied to the cell in step 3032 to increment the value of local variable TRE in step 3034 in preparation for attempting to re-establish the DNA-polymerase mechanical change-sensor component within the porin, beginning in step 3018. If the value stored in local variable TRE is greater than or equal to the value of parameter param[retry] determined in step 3030, then an indication of failure is returned by the routine "configure sequencer".

[0089] FIG. 30C provides a control-flow diagram of the routine called "read array" in step 3010 of FIG. 30A. The routine "read array" represents the execution of the sequence determination performed by the sequence detection system. The routine "read array" repeats a series of voltage-application cycles until a termination condition occurs. In step 3040, a memory buffer represented by the array currentData is initialized with an index nxtSample indicating the next location in the memory buffer to store the signal sample. In step 3042, the routine "read array" applies a voltage of magnitude param[read volts] and polarity parameter [read Pol] to the cell for a length of time indicated by parameter buffer [read interval] and collects the sampled output signal in memory buffer currentData. In step 3044, the routine "read array" then applies a voltage of magnitude param[reverse voltage] and polarity param[reverse Pol] to the cell for a length of time indicated by parameter param[reverse interval]. This reverse polarity voltage application suppresses the generation of sequence information masking noise, as discussed above with reference to FIG. 25. In step 3046, the routine "read array" checks for a termination event by calling the routine "check for termination." If a termination event occurs, as determined in step 3048, the routine "read array" returns. Otherwise, control returns to step 3042 for another voltage application cycle.

[0090] FIG. 30D provides a control-flow diagram for the routine "check done" called in step 3046 of FIG. 30D. In step 3050, the routine "check done" determines whether a power off event has occurred, indicating that the cell is about to be powered down. If so, the routine "check done" returns a finish indication. Otherwise, in step 3052, "check done" analyzes the most recent output signal sample stored in the memory buffer currentData. The most recent output signal sample is indicated by the parameter value param[window], and uses the array data with a number of the most recent output signal samples in the memory buffer currentData to determine whether data is still being generated by the cell at a rate that exceeds the threshold sequence data rate. If the analysis indicates that the array data is still being collected at an acceptable rate, as determined in step 3054, the routine "check done" returns an indication to continue. Otherwise, the latest routine "check done" returns a finish indication. The rate of array data may decrease due to clogging of the porin channels, deterioration of the electrodes, and many other reasons.

[0091] Although the present invention has been described with respect to specific embodiments, it is not intended to limit the present invention to these embodiments. Modifications within the spirit of the present invention will be apparent to those skilled in the art. For example, a particular sequence detection system can use a variety of different types of mechanical change sensor components, mechanical couplers, variable resistance components, electrical signal generation components and circuits. For example, a particular sequence detection system can use various types of mechanical change sensor components, mechanical couplers, variable resistance components, electrical signal generation 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 Neisserial 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 aquatic Pol I, Bacillus stearothermophilus Pol, 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, Pol lambda, 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. Another example of a tether includes synthetic polymers such as PEG (polyethylene glycol), PPG (polypropylene glycol), PVA (polyvinyl alcohol), and PE (polyethylene). Additionally, include, for example, DNA, RNA, PNA or LNA labels, morpholino, or chiral DNA. Additionally, in one exemplary embodiment, isoG and isoC bases can be present on the tether. A variety of different values ​​derived from the output voltage signal can be used as different aspects to uniquely identify the type of object or entity in the target sequence. The magnitude, variance, and standard deviation of the average voltage signal calculated from the voltage signal are just a few examples of the various types of derived values ​​that can be used to identify the type of object or entity. Many different types of modified deoxynucleotide triphosphates can be used for copy strand extension in addition to 7-deaza deoxyadenosine triphosphate.Because copy strand extension is used solely to determine the sequence of the template strand, the chemical composition of the copy strand generated during sequencing is irrelevant to the successful operation of the second sequence detection system. All combinations of the concepts, components, and methods discussed are contemplated and understood.

[0092] 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-signal transducer that converts one or more mechanical changes into a signal; an analysis subsystem that uses the signals to determine a sequence of types of entities within the target; and initializing the sequence-detection system according to one or more operational parameters; and Sequencing - Controlling a detection system to generate signals, collect samples of the signals, and analyze the signals to determine a sequence of types of entities within the target. Control Subsystem Including, The target is a primer-associated nucleic acid template polymer; The entity is a nucleotide monomer; the mechanical change sensor is a nucleic acid polymerase contained within a chamber containing a first solution; and Mechanical change – signal transducers also a variable resistor that regulates the flow of current through a porin channel that provides fluid communication between a chamber containing a first solution and a chamber containing a second solution; a mechanical coupler coupling the mechanically variable component to the variable resistor; and a current-to-voltage converter that outputs a voltage signal that varies in response to a rate of current flow through the porin channel; The operating parameters are: the voltage magnitude of the read voltage applied across the porin channel and the polarity of the read voltage; the voltage magnitude of the noise-blocking voltage applied across the porin channel and the polarity of the noise-blocking voltage; The magnitude of the constitutive voltage applied across the porin channel and the polarity of the constitutive voltage Including, Sequence detection system.

2. the polarity of the noise blocking voltage is opposite to the polarity of the read voltage; and The sequence detection system of claim 1 , wherein the polarity of the noise-blocking voltage is opposite to the polarity of the constituent voltages.

3. 2. The sequence detection system of claim 1, wherein the control subsystem configures the sequence detection system by applying a constitutive voltage with a constitutive polarity across the porin channel to seat the nucleic acid polymerase mechanical change sensor within the porin.

4. The sequence detection system of claim 3 , wherein following configuration of the sequence detection system, the control subsystem adjusts the magnitude of the read voltage to position the reporter region of the nucleic acid polymer tether at the reference displacement.

5. Following configuration of the sequence detection system, the control subsystem controls the sequence detection system to: applying a read voltage across the porin channel at a read voltage polarity for a first time period during which the voltage output signal is sampled, stored in memory, and analyzed by an analysis subsystem; and applying a noise-inhibiting voltage across the porin channel for a second time period with a polarity of the noise-inhibiting voltage; The sequence detection system of claim 3, which generates signals, collects samples of the signals, and analyzes them to determine a sequence of types of entities within the target by repeating this process until an end condition occurs.

6. The sequence detection system of claim 5 , wherein the first and second time periods are specified by an operational parameter.

7. The termination condition is Power off events; and Reducing the rate at which sequence detection systems generate sequence information below a threshold level The sequence detection system of claim 5 .

8. The operating parameters are: temperature; the pH of the solutions in the chambers containing the first and second solutions; and Contents of the solutions in the chambers containing the first and second solutions The sequence detection system of claim 1 .

9. The operating parameters related to the contents of the solutions in the chambers containing the first and second solutions are: One or more types of ionized salts in solution; The concentration of one or more types of ionized salts in the solution; one or more types of nucleotide polyphosphates in solution in a chamber containing a first solution; a concentration of one or more types of nucleotide polyphosphates in a solution in the chamber containing the first solution; one or more types of primer-associated nucleic acid template polymers in solution in a chamber containing a first solution; a concentration of one or more types of primer-associated nucleic acid template polymers in a solution in the chamber containing the first solution; and Nucleic Acid Polymer Tether Types and Structures The sequence detection system according to claim 8 , comprising one or more of the following:

10. 9. The sequence detection system of claim 8, wherein an operational parameter associated with the contents of the solution in the chamber containing the first solution is adjusted to control the rate of sequential specific interactions between entities in the target and the mechanical change sensor.

11. 10. The sequence detection system of claim 8, wherein a primer having a 3' dideoxynucleotide monomer is used to restrict the sequence detection system to specifically interact with a single entity.

12. 2. The sequence detection system of claim 1, wherein the signal response of the sequence detection system is constructed by selecting a reporter region having a nucleotide-monomer sequence that corresponds to the signal response.

13. The sequence detection system of claim 1, wherein the nucleic acid polymerase is the Klenow fragment of DNA polymerase I.

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