WO2013065881A1

WO2013065881A1 - Apparatus and method for determining sequences of nucleic acids using atomic force microscope

Info

Publication number: WO2013065881A1
Application number: PCT/KR2011/008200
Authority: WO
Inventors: Bongchu Shim; Jeankun Oh; Jitae Kim; Dami Kim; Yong Shin; Seongmoon Cho
Original assignee: LG Electronics Inc
Current assignee: LG Electronics Inc
Priority date: 2011-10-31
Filing date: 2011-10-31
Publication date: 2013-05-10
Anticipated expiration: 2014-04-30

Abstract

The present invention relates to an apparatus for determining sequences of nucleic acids using AFM (Atomic Force Microscope) capable of increasing a polymerization speed of a target nucleic acid molecule and enhancing accuracy of analysis through polymerization reaction, and a method using the apparatus, where the apparatus includes a polymerase attached to a tip of a cantilever for the AFM, and a substrate attached by a dNTP, and where the polymerase is moved on a substrate.

Description

APPARATUS AND METHOD FOR DETERMINING SEQUENCES OF NUCLEIC ACIDS USING ATOMIC FORCE MICROSCOPE

The teachings in accordance with exemplary embodiments of this invention relate generally to an apparatus and method for determining sequences of a target nucleic acid molecule, and more particularly to an apparatus for determining sequences of nucleic acids using atomic force microscope capable of controlling a polymerization speed of a target nucleic acid molecule and increasing accuracy of analysis through polymerization reaction, and a method using the apparatus.

Many attempts have been waged to analyze sequences of target nucleic acids molecules using a variety of techniques. Among the techniques, a conventional analysis technique using an optical method enables analysis of the sequences only through replication for signal amplification. Furthermore, reagent, in which DNA replication process and fluorescent luminary are bound, is basically required as expendables.

Thus, recently, a next generation sequencing technique is being studied that is capable of analyzing sequences using only ssDNA free from replication. The DNA replication is a method that removes waste of expenses including time and reagent consumption and sequentially reads sequences in a process in which ssDNA is synthesized using polymerase.

An AFM (Atomic Force Microscope) has been used as an important means capable of understanding various reaction mechanisms among bio-molecules in a body. The AFM, being capable of analyzing bonding among bio-molecules, is expected to be efficiently used in future nano and biotechnical fields including various molecular researches.

In a non-limiting example, a nano mechanical device such as a micro cantilever enables characteristic analysis of interactions in various biological molecules such as DNA polymerization, protein Ag-Ab binding, RNA-protein interaction, peptide-drug interaction and ligand-binding on membrane protein. The label-free detection in the interaction is typically based on cantilever bending deflection change measurement generated from the molecular interactions.

Importance of researches on bio-molecules using the bio-AFM technique is rapidly on the increase. The bio-AFM technique is a very important tool in studying structures and substructures of bio-molecules because the bio-AFM technique can observe in a nano meter unit nonconductive materials such as bio-molecules under liquefied condition assisting biological activation. Furthermore, the bio-AFM, being capable of closely observing bindings of molecules using its bio-AFM tip, can load bio-molecules, and high sensitivity bio-AFM is initially important because the bio-AFM is applied to measure binding of complementary DNA molecules, binding of proteins, ligand-reception binding which is important in studying immune response and binding in bio-molecules in a single molecule.

However, the conventional art using the bio-AFM is disadvantageous in that all the dNTPs are melted in solution to reduce a yield of polymerization of target nucleic acid molecules and an analysis speed resultant therefrom. Furthermore, in order to study mechanism of target nucleic acid molecules, ligand must be attached to a tip of an AFM, and according to the conventional method using a biotin-streptavidin binding or a synthetic self-assembly film, there is no way to directly adjust a distance between molecules, and ligands may be concentrated to a particular portion, making it difficult to accurately measure ligand-receptor binding.

The present invention is disclosed to solve the aforementioned disadvantages and/or problems and it is an object of the present invention to determine sequences of target nucleic acid molecules using a cantilever for AFM while controlling a polymerization speed of the target nucleic acid molecules. Furthermore, it is another object to provide an apparatus for determining sequences having a high analytic accuracy by the polymerization reaction and a method thereof.

Technical problems to be solved by the present invention are not restricted to the above-mentioned, and any other technical problems not mentioned so far will be clearly appreciated from the following description by skilled in the art.

An object of the invention is to solve at least one or more of the above problems and/or disadvantage in whole or in part and to provide at least the advantages described hereinafter. In order to achieve at least the above objects, in whole or in part, and in accordance with the purposes of the invention, as embodied and broadly described, and in one general aspect of the present invention, there is provided an apparatus for determining sequences of nucleic acids using an AFM (Atomic Force Microscope), the apparatus characterized by: polymerase attached to a tip of a cantilever for AFM; a primer attachable to a single stranded target nucleic acid molecule polymerized by the polymerase; a substrate attached per type by dNTP hybridized with the single stranded target nucleic acid molecule; and moving means moving the polymerase on the substrate.

In some exemplary embodiments, the apparatus is further characterized by a dendron molecule attaching the polymerase to the tip of the cantilever.

In some exemplary embodiments, the primer is a universal primer.

In some exemplary embodiments, the dNTP is arranged in parallel on the substrate for each type.

In some exemplary embodiments, the dNTP includes a plurality of dNTP sets formed with different types of dNTPs.

In some exemplary embodiments, the dNTP is such that an outermost phosphate is fixed on the substrate.

In some exemplary embodiments, the tip of the cantilever is charged with a positive polarity.

In some exemplary embodiments, the moving means repetitively scan the dNTP per type.

In some exemplary embodiments, the apparatus is further characterized by a controller measuring in real time changes in force generated from the tip of the cantilever by the polymerization.

In some exemplary embodiments, the controller changes a path of the moving means in response to a changed value of the measured force.

In another general aspect of the present invention, there is provide a method for determining sequences of nucleic acids using an AFM (Atomic Force Microscope), the method characterized by: attaching polymerase to a tip of a cantilever for AFM; connecting a single stranded target nucleic acid molecule attached with a primer to the polymerase; and moving the polymerase connected with the single stranded target nucleic acid molecule on a substrate attached by dNTP per type.

In some exemplary embodiments, the step of attaching polymerase to a tip of a cantilever for AFM includes connecting dendron molecule to the tip of the cantilever, and attaching the polymerase to the dendron molecule.

In some exemplary embodiments, the step of connecting a single stranded target nucleic acid molecule to the polymerase includes; attaching a primer to the single stranded target nucleic acid molecule; and connecting the single stranded target nucleic acid molecule attached with the primer to the polymerase.

In some exemplary embodiments, the substrate is arranged in parallel with dNTP per type.

In some exemplary embodiments, the step of moving the polymerase on the substrate includes repetitively moving the polymerase on the substrate for each type of dNTP.

In some exemplary embodiments, the method is further characterized by applying (+) bias to the tip of the cantilever.

In some exemplary embodiments, the method is further characterized by measuring in real time changes in force generated from the tip of the cantilever by polymerization of the single stranded target nucleic acid molecule and the dNTP.

In some exemplary embodiments, the method is further characterized by moving the polymerase to another path in response to the changed value of the measured force.

The present invention has an advantageous effect in that polymerase attached to a tip of a cantilever for AFM is used, and the polymerase is reacted with dNTP fixed to a substrate to control a polymerization speed of target nucleic acid molecule.

The present invention has another advantageous effect in that the polymerization reaction occurs at a predetermined height on the substrate to greatly increase accuracy of analysis due to no limit in read length.

The present invention has still another advantageous effect in that a user can control a moving speed of a tip attached with the polymerase to adjust a speed of the polymerization reaction as desired, whereby the sequences can be analyzed more accurately.

Various aspects and embodiments of the invention are described in further detail below.

The technical solution is neither intended nor should it be construed as being representative of the full extent and scope of the present invention, which these and additional aspects will become more readily apparent from the detailed description, particularly when taken together with the appended drawings. As mentioned above, the technical solution is not an extensive overview and is not intended to identify key or critical elements of the apparatuses, methods, systems, processes, and the like, or to delineate the scope of such elements. The technical solution provides a conceptual introduction in a simplified form as a prelude to the more-detailed description that follows.

The teachings of the present invention can be readily understood by considering the following detailed description in conjunction with the accompanying drawings, in which:

FIG.1 is a mimetic diagram illustrating an apparatus for determining sequences of nucleic acids using an AFM (Atomic Force Microscope) according to an exemplary embodiment of the present invention;

FIG.2 is an enlarged view of a state in which polymerase is attached to a tip of a cantilever through dendron according to an exemplary embodiment of the present invention;

FIG.3 is a mimetic diagram illustrating polymerase moving on a substrate according to an exemplary embodiment of the present invention;

FIG.4 is a mimetic diagram illustrating a state of dNTP set being arranged on a substrate according to an exemplary embodiment of the present invention;

FIG.5 is a mimetic diagram illustrating a state of dNTP arranged in a round shape on a substrate according to an exemplary embodiment of the present invention;

FIG.6 is a graph illustrating a change in force measured from a tip of a cantilever according to an exemplary embodiment of the present invention; and

FIG.7 is a mimetic diagram illustrating polymerase moving on a substrate to a changed path according to an exemplary embodiment of the present invention.

The following description is not intended to limit the invention to the form disclosed herein. Consequently, variations and modifications commensurate with the following teachings, and skill and knowledge of the relevant art are within the scope of the present invention. The embodiments described herein are further intended to explain modes known of practicing the invention and to enable others skilled in the art to utilize the invention in such, or other embodiments and with various modifications required by the particular application(s) or use(s) of the present invention.

The disclosed embodiments and advantages thereof are best understood by referring to FIGS. 1-7 of the drawings, like numerals being used for like and corresponding parts of the various drawings. Other features and advantages of the disclosed embodiments will be or will become apparent to one of ordinary skill in the art upon examination of the following figures and detailed description. It is intended that all such additional features and advantages be included within the scope of the disclosed embodiments, and protected by the accompanying drawings. Further, the illustrated figures are only exemplary and not intended to assert or imply any limitation with regard to the environment, architecture, or process in which different embodiments may be implemented. Accordingly, the described aspect is intended to embrace all such alterations, modifications, and variations that fall within the scope and novel idea of the present invention.

Aspects of the present teachings may be further understood in light of the following examples, which should not be construed as limiting the scope of the present teachings in any way. The section headings used herein are for organizational purposes only and are not to be construed as limiting the described subject matter in any way. All literature and similar materials cited in this application, including but not limited to, patents, patent applications, articles, books, treatises, and internet web pages are expressly incorporated by reference in their entirety for any purpose. When definitions of terms in incorporated references appear to differ from the definitions provided in the present teachings, the definition provided in the present teachings shall control. It will be appreciated that there is an implied "about" prior to the temperatures, concentrations, times, etc discussed in the present teachings, such that slight and insubstantial deviations are within the scope of the present teachings herein. In this application, the use of the singular includes the plural unless specifically stated otherwise. For example, "a primer" means that more than one primer can, but need not, be present; for example but without limitation, one or more copies of a particular primer species, as well as one or more versions of a particular primer type, for example but not limited to, a multiplicity of different forward primers. Also, the use of "comprise", "comprises", "comprising", "contain", "contains", "containing", "include", "includes", and "including" are not intended to be limiting. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention.

That is, the terms "including", "includes", "having", "has", "with", or variants thereof are used in the detailed description and/or the claims to denote non-exhaustive inclusion in a manner similar to the term "comprising".

Furthermore, "exemplary" is merely meant to mean an example, rather than the best. It is also to be appreciated that features, layers and/or elements depicted herein are illustrated with particular dimensions and/or orientations relative to one another for purposes of simplicity and ease of understanding, and that the actual dimensions and/or orientations may differ substantially from that illustrated. That is, in the drawings, the size and relative sizes of layers, regions and/or other elements may be exaggerated or reduced for clarity. Like numbers refer to like elements throughout and explanations that duplicate one another will be omitted. Now, the present invention will be described in detail with reference to the accompanying drawings.

Words such as "thereafter," "then," "next," etc. are not intended to limit the order of the processes; these words are simply used to guide the reader through the description of the methods.

It will be understood that when an element is referred to as being "connected" or "coupled" to another element, it can be directly connected or coupled to the other elements or intervening elements may be present. In contrast, when an element is referred to as being "directly connected" or "directly coupled" to another element, there are no intervening elements present. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items and may be abbreviated as "/".

It will be understood that, although the terms first, second, etc. may be used herein to describe various elements, these elements should not be limited by these terms. These terms are only used to distinguish one element from another. For example, a first region/layer could be termed a second region/layer, and, similarly, a second region/layer could be termed a first region/layer without departing from the teachings of the disclosure.

The following terms, as used in the present specification and claims, are intended to have the meaning as defined below, unless indicated otherwise

As used herein, the term of "nucleic acid" may refer to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides. Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral methyl phosphonates, 2-O-methyl ribonucleotides, and peptide-nucleic acids (PNAs). The nucleic acid may be DNA, RNA or any naturally occurring or synthetic modification thereof, and combinations thereof. Preferably however the nucleic acid will be DNA, which may be genomic, or, cDNA, and single or double stranded or in any other form.

As used herein, the nucleic acid being sequenced may be referred to as the target nucleic acid (or the target). Target nucleic acids include but are not limited to DNA such as but not limited to genomic DNA, mitochondrial DNA, cDNA and the like, and RNA such as but not limited to mRNA, miRNA, and the like. That is, the term "target nucleic acid" may refer to a polynucleotide sequence that is sought to be amplified and/or quantified. The target polynucleotide can be obtained from any source, and can comprise any number of different compositional components. For example, the target can be nucleic acid (e.g. DNA or RNA), transfer RNA, sRNA, and can comprise nucleic acid analogs or other nucleic acid mimic, though typically the target will be messenger RNA (mRNA) and/or micro RNA (miRNA).

As used herein, the term " polymerization" may refer to the complementary base-pairing interaction of one nucleic acid with another nucleic acid that results in the formation of a duplex, triplex, or other higher-ordered structure, and is used herein interchangeably with "annealing."

As used herein, the term "detection" may refer to any of a variety of ways of determining the presence and/or quantity and/or identity of a target polynucleotide.

As used herein, the term "nucleic acid molecule" may refer to a sequence of contiguous nucleotides (riboNTPs, dNTPs or ddNTPs, or combinations thereof) of any length which can encode a full-length polypeptide or a fragment of any length thereof, or which can be non-coding. As used herein, the terms "nucleic acid molecule" and "polynucleotide" can be used interchangeably and include both RNA and DNA

As used herein, the term "target" may refer to a range of molecules including but not limited to a miRNA (or pre-miRNAs), an siRNA, a piRNA, a long non-coding RNA, an mRNA, rRNA, tRNA, hnRNA, cDNA, genomic DNA, and long noncoding RNA (ncRNA).

As used herein, the term "complementary" and "complementarity" are interchangeable and may refer to the ability of polynucleotides to form base pairs with one another. Base pairs are typically formed by hydrogen bonds between nucleotide units in antiparallel polynucleotide strands or regions.

As used herein, the term "ligand" may refer to something which binds.

As used herein, the term “dNTP (deoxy Nucleoside Tree)” may refer to a deoxy-nucleotide tri-phosphate or a deoxyribonucleotide, and may include nucleotide analog), and may be dATP, dCTP, dGTP, dTTP, and dUTP. The deoxyribonucleotide is the monomer, or single unit of DNA, or deoxyribonucleic acid. Each deoxyribonucleotide comprises three parts: a nitrogenous base, a deoxyribose sugar, and one phosphate group.

A dendron which is coated on the surface of substrate in this disclosure is a kind of dendrimer. Dendrimers are highly branched polymers with uniform size and molecular weight as well as a well-defined structure. They consist of a central multifunctional core, multifunctional repeating unit attached around the core, and a terminal or end group. According to their shape, they are divided into two types. The first one has a circular or elliptic shape of which repeating units are regularly stretched from a core, whereas the second type has a conic shape of which repeating units are directionally stretched from a core. The second type is generally called as dendron.

Unless defined otherwise, technical terms used herein have the same meaning as commonly understood by those skilled in the art or in definitions found in technical literature, e.g., Dictionary of Microbiology and Molecular Biology, 2nd ed. (Singleton et al., 1994, John Wiley & Sons, New York, N.Y.), The Harper Collins Dictionary of Biology (Hale & Marham, 1991, Harper Perennial, New York, N.Y.), and similar publications. Unless described otherwise, techniques employed or contemplated herein are standard well known methods.

FIG.1 is a mimetic diagram illustrating an apparatus for determining sequences of nucleic acids using an AFM (Atomic Force Microscope) according to an exemplary embodiment of the present invention, FIG.2 is an enlarged view of a state in which polymerase is attached to a tip of a cantilever through dendron according to an exemplary embodiment of the present invention and FIG.3 is a mimetic diagram illustrating polymerase moving on a substrate according to an exemplary embodiment of the present invention.

The apparatus for determining sequences of nucleic acids using an AFM (Atomic Force Microscope) includes polymerase (130) attached to a tip (120) of a cantilever (110) for AFM; a primer (200) attachable to a single strand of target nucleic acid molecule (10) (or a single strand of target nucleic acid molecule (10), which is interchangeably used herein), polymerized by the polymerase; a substrate (300) attached per kind by dNTP hybridized with the single strand of target nucleic acid molecule; and moving means (400) moving the polymerase (130) on the substrate.

The present invention includes a polymerase (130) attached to a tip (120) of a cantilever (110) for AFM, and a substrate (300) attached per kind by dNTP hybridized with the single strand of target nucleic acid molecule (10), where the polymerase (130) is moved on the substrate (300), and the single strand of target nucleic acid molecule (10) is hybridized.

To be more specific, the polymerase (130) is attached to the tip (120) of the cantilever (110) of AFM. In the present invention, the polymerase (130) is attached to the tip (120) of the cantilever (110) of AFM, whereby the polymerization of target nucleic acid molecule by the polymerase can be measured in real time through changes of force generated from the tip (120).

The polymerase (130) is formed by polymerizing a single strand of target nucleic acid molecule (10) by two strands, and may be DNA polymerase or RNA polymerase. The types, sizes or shapes of the polymerase (130) are not particularly limited. A method of attaching the polymerase (130) to the tip of the cantilever of the AFM is not particularly limited either, and the method may include all known arts in the related fields. For example, the method may use a biotin-streptavidin binding or a synthetic self-assembly film.

Preferably, as shown in FIG.2, in a case a dendron molecule (121) is connected to the tip (1200 of the cantilever (110), the polymerase (130) is attached to the dendron molecule (121), and more polymerases can be accurately attached to a target portion. Furthermore, quantity and position of the polymerase (130) can be controlled by connecting a single strand of nucleic acid molecule (122) to a tip of the dendron molecule (121).

Thus, the apparatus for determining sequences of nucleic acids using an AFM (Atomic Force Microscope) according to the present invention may further include a dendron molecule attaching the polymerase (130) to the tip (120) of the cantilever (110).

The primer (200) may be attached to a single strand of target nucleic acid molecule (10) polymerized by the polymerase (130). The single strand of target nucleic acid molecule (10) cannot recognize the polymerase (130) by itself, such that the present invention can connect the single strand of target nucleic acid molecule (10) using the primer (200).

To this end, the primer (200), the primer is preferably a universal primer. For example, a forward primer (200) is attached to the single strand of target nucleic acid molecule (10) using legation, and the forward primer (200) is attached to a reverse primer complementary thereto, to make a portion of the single strand of target nucleic acid molecule (10) a double strand. Furthermore, it is also possible to connect the primer to a particular portion of the single strand of target nucleic acid molecule (10) that is to be analyzed.

The substrate (300) is attached by dNTP (310) per kind hybridized with the single strand of target nucleic acid molecule (10). The present invention is characterized by the dNTP being attached in parallel on the substrate (300), and particularly by the dNTP (310) being attached on the substrate (300) for each type. Therefore, the present invention is characterized in that the polymerase (130) is moved on the substrate (300) to hybridize the single strand of target nucleic acid molecule (10).

To this end, the substrate (300) may include a conductive material or a non-conductive material based on purpose of use according to the present apparatus, and may include any one from a group consisting of silver, copper, gold, aluminum, glass, ceramic, silicon, Si/SiO2 wafer, polystyrene, polyethylene, polypropylene and polyacrylamide, but the material is not limited thereto.

Furthermore, a surface of the substrate (300) may include gold for easily attaching the dNTP (310), a gold layer may be formed on a silicone nitrade, and the surface of the substrate (300) may be modified to have ?COOH, -SH, -OH, silan group, amin group or epoxy group. The substrate may take any shape as long as the dNTP (310) can be fixed thereto, but it is preferable that the substrate take a shape of a plate.

The dNTP (310) may include a plurality of dCTP (311), dGTP (312), dTTP (313) and dATP (314), and these d NTPs are preferably divided per type and attached to the surface of the substrate (300). For example, as depicted in FIG.1, the dNTP is preferably arranged in parallel on the substrate for each type.

That is, the dCTP (311), dGTP (312), dTTP (313) and dATP (314) are arranged in four rows, and each dNTP (310) may form a line pattern attached in a row from each line. Thus, the present invention can accurately anticipate the sequence polymerized by the polymerase (130).

It would be sufficient that the dCTP (311), dGTP (312), dTTP (313) and dATP (314) are mutually distinguished, and each attached position, shape or order is not particularly limited.

FIG.4 is a mimetic diagram illustrating a state of dNTP set being arranged on a substrate according to an exemplary embodiment of the present invention, where as shown in the drawing, the dNTP (310) includes a plurality of dNTP sets (310a, 310b) formed with different kinds of dNTPs (311a, 312a, 313a, 314a), and as shown in FIG.5, the dNTP (310) may be formed in a circularly arranged pattern.

In an exemplary embodiment, the dNTP (310), in being fixed to the substrate (300), is preferably such that an outermost phosphate is fixed on the substrate (300). That is, a third phosphate of the dNTP (310) may be fixed to the substrate (300), or a second phosphate of the dNTP (310) may be fixed to the substrate (300).

The dNTP (310) structurally includes one phosphate group, a nitrogenous base, and a deoxyribose sugar, and among these, two phosphates are detached from one phosphate group along with the nitrogenous base, and the deoxyribose sugar by the polymerization of the polymerase (130). Thus, the dNTP(310) is easy to allow the two phosphates to be separated, the outermost phosphate being fixed to the substrate (300).

A method of attaching the dNTP(310) to the substrate(300) is not particularly limited either. The abovementioned attaching method of polymerase (130) may be used, and amine group positioned at a tip of a molecule having nanocone may be used for attachment, for example.

The moving means (not shown) is intended to move the polymerase (130) on the substrate (300). It is sufficient enough for the moving means to move the polymerase (130) coupled by the single strand of target nucleic acid molecule (10), and it is also possible for the moving means to move the tip (120) including the cantilever (130), the cantilever (130) or the AFM apparatus. For example, moving means of AFM apparatus forming a membrane pattern in the conventional semiconductor manufacturing apparatus may be applied.

Referring to FIG.3 again, the substrate (300) is attached with the dNTP (310) per type, and in the present invention, the types of single strand of target nucleic acid molecule (10) reacting with the polymerase (130) can be known by controlling a path (400) of the moving means. As illustrated in FIG.4, the moving means repetitively scan single strand of target nucleic acid molecule (10) per type, and as shown in FIG.5, it is also possible for the moving means to make a circle from a center to move to the outside.

Thus, the present invention is such that the polymerase (130) recognizes and captures a single strand of target nucleic acid molecule (10) to be analyzed, the recognized and captured single strand of target nucleic acid molecule (10) is moved to the substrate (300), where the polymerase (130) is positioned at a distance proximate to the single strand of target nucleic acid molecule (10). Then, the polymerase (130) polymerizes the single strand of target nucleic acid molecule (10) and the dNTP (310).

Successively, in a case the polymerase (130) is continuously moved along a designated path, the dNTP (310) corresponding to the single strand of target nucleic acid molecule (10) is reacted to realize the polymerization. At this time, changes in force generated from the tip (120) of the cantilever (110) may be measured to determine the sequence. As a result, the present invention may further include a controller measuring in real time the changes in force generated from the tip (120) of the cantilever (110) by the polymerization.

In the exemplary embodiment, the tip (120) of the cantilever (110) is preferably charged with a positive polarity. In a case the polymerase (130) attached to the tip (120) of the cantilever (110) recognizes and captures a single strand of target nucleic acid molecule (10), and the tip (120) is charged with the positive polarity, the dNTP (310) charged with a negative polarity can be more easily attracted.

For example, the tip (120) of the cantilever (110) is charged with a positive (+) bias to allow the dNTP (310) to be electrically attracted and collected at the tip (120), whereby efficiency of polymerization can be enhanced. To this end, the present invention may further include a power supply unit for supplying voltage to the cantilever (110) or the tip (120) of the cantilever (110).

Furthermore, the apparatus for determining sequences of nucleic acids using an AFM (Atomic Force Microscope) according to the present invention may be applied with prior art except for the polymerase (130), the primer (200), the substrate (300) and the moving means (400), the configuration of which is different from the prior art.

That is, the apparatus for determining sequences of nucleic acids using an AFM (Atomic Force Microscope) according to the present invention may further include a laser generating unit (140) irradiating laser (141) to the cantilever (110), a laser sensor (142) receiving laser reflected from the cantilever (110), a sensor signal processor (150) processing a signal received by the laser sensor (142), and an AFM controller (160) controlling the sensor signal processor (150). Furthermore, the apparatus may still further include a cantilever controller (170) controlling the cantilever (110).

In this case, the laser sensor (142) may be an optical detector or an electric detector. The detector used for electric detection may be to detect one or more from a group consisting of a current, a voltage, a resistance and an impedance, for example, and the detector for optical detection may be to detect one or more of a group consisting of light absorption, light transmission, fluorescent light, FRET (Fluorescence Resonance Energy Transfer), surface plasmon resonance, SERS (Surface-Enhanced Raman Scattering) and diffraction.

Furthermore, albeit not being described in detail, the apparatus may form each functional element with a process-on-a-chip or a lab-on-a-chip using known microfluidic units and MEMS (micro electro mechanical systems) device.

Hereinafter, a an apparatus for determining sequences of a target nucleic acid molecule using an AFM (Atomic Force Microscope) using an atomic force microscope will be described with reference to FIGS. 1 to 7.

The method, as noted from the foregoing, may be performed using an apparatus for determining sequences of a target nucleic acid molecule using an atomic force microscope including a polymerase (130), a primer (200), a substrate (300) and moving means (400).

First of all, the method for determining sequences of a target nucleic acid molecule using an AFM includes attaching polymerase (130) to a tip (120) of a cantilever (110) for AFM (S100). The method of attaching polymerase (130) to a tip (120) of a cantilever (110) for AFM is not particularly limited.

For example, in attaching polymerase (130) to a tip (120) of a cantilever (110) for AFM, analytic accuracy can be further enhanced by accurately attaching more polymerases (130), in case a dendron molecule (121) is connected to the tip (120) of the cantilever (110) and the polymerase (130) is attached to the dendron molecule (121).

Furthermore, the method includes connecting a single stranded target nucleic acid molecule (10) attached with a primer (200) to the polymerase (130) (S200).

The primer (200) and the target nucleic acid molecule (10) attached with the primer has been already explained in the above, where although an order of attaching the primer (200) to the single stranded target nucleic acid molecule (10) is not particularly restricted, it is preferable that the primer (200) be attached to the single stranded target nucleic acid molecule (10), and the single stranded target nucleic acid molecule (10) attached with the primer (200) be connected to the polymerase (130), whereby the polymerase (130) is such that the analysis-objected single stranded target nucleic acid molecule (10) is recognized and captured by the primer (200), where the polymerase (130) and the single stranded target nucleic acid molecule (10) are mutually connected.

At this time, the step (S100) of attaching the polymerase (130) and the step (S200) of connecting the single stranded target nucleic acid molecule (10) may be separately realized, but may be realized at one time. That is, the tip (120) of the cantilever (110) is made to be in a state in which the polymerase (130) can be attached, where a buffer including the polymerase (130), the single stranded target nucleic acid molecule (10), and the primer (200) may be reacted.

Successively, the method according to the present invention may include moving the polymerase (130) connected with the single stranded target nucleic acid molecule (10) on a substrate (300) attached by dNTP (310) per type (S300). The substrate (300) attached by dNTP (310) per type has been already explained in the apparatus section, such that the dNTP (310) is preferably arranged in parallel per type on the substrate (300), as shown in FIG.3.

At this time, the step of attaching the dNTP (310) to the substrate (300) may be such that a state in which the dNTP (310) can be attached to a particular position of the substrate (300) is pre-made, and the buffer further including the dNTP (310) is utilized. That is, the tip (120) of the cantilever (110) is prepared for a state attachable with the polymerase, a particular position of the substrate (300) is prepared for a state attachable with the dNTP (310), and then the buffer including the polymerase (130), the single stranded target nucleic acid molecule (10), and the primer (200) is reacted.

The buffer may further include other reactive additives for promoting the coupling between the single stranded target nucleic acid molecule (10) and the dNTP (310). For example, the buffer may further include Mg2 or calcium chloride. Then, a condition in which the single stranded target nucleic acid molecule (10) and the dNTP (310) are coupled by the polymerase (130) is prepared about the substrate (300).

Thereafter, the polymerase (130) is sequentially moved on the substrate (300) attached by the polymerase (130) is sequentially moved per type. The step of moving the polymerase (130) on the substrate preferably includes repetitively moving the polymerase (130) on the substrate for each type of dNTP.

In a case the polymerase (130) is moved to meet a dNTP (310) coupleable with the single stranded target nucleic acid molecule (10), the polymerase (130) cuts a second rear phosphate of triphosphate group of the dNTP (310) to complete the polymerization.

There occurs a change in force on the tip (120) of the cantilever (110) by the polymerization, and the sequence is determined by measuring the change in force. Therefore, the method is further characterized by measuring in real time changes in force generated from the tip of the cantilever by polymerization of the single stranded target nucleic acid molecule and the dNTP (S400).

For example, the base of the dNTP (310) includes adenine, guanine, cytosine and thymine, each base being different in chemical structure, different changes by forces through the polymerization can be generated and differences are detected by electrical signals whereby the sequences of DNA can be analyzed. Furthermore, a user can arrange the dNTP (310) on a particular desired position of the substrate (300) and can control a moving path of the polymerase (130). As a result, it is also possible to determine the sequences of DNA, not by differences in types of bases of the dNTP (310), but by differences in signals resultant from the coupling.

For example, as shown in FIG.3, in a case the single stranded target nucleic acid molecule (10) and a complementary base are recruited by the polymerase (130) on the dNTP substrate (300), a force generated by coupling between the single stranded target nucleic acid molecule (10) and the dNTP (310) is transmitted to the tip (120) of the AFM, whereby change of laser is detected by a sensor (142), and the change is recorded by a sensor signal processor (150). The recorded signal is transmitted to the AFM controller (160) to determine the sequences.

At the same time, the method according to the present invention may further include applying (+) bias to the tip (120) of the cantilever (110) before and after the polymerase (130) is moved or when the polymerase (130) is moved. As a result, the tip (120) is charged with a positive charge to easily and electrically attract the dNTP (310) charged with a negative charge.

Meantime, FIG.6 is a graph illustrating a change in force measured from a tip of a cantilever according to an exemplary embodiment of the present invention, and FIG.7 is a mimetic diagram illustrating polymerase moving on a substrate to a changed path according to an exemplary embodiment of the present invention.

Referring to FIG.6, changes in force may be differently shown depending on base types (A, T, G, C) of the dNTP (310), and the present invention can determine the sequences by the differences.

Furthermore, in a case analysis-objected single stranded target nucleic acid molecule (10) has a continuous sequence (e.g., GGG), a signal determining a first sequence in the continuous sequence may be differently shown from that of a non-continuous sequence (e.g., G) by the next dNTP (310) that is adjacently attached per type and magnesium ion existing thereabout. That is, a signal of right hand side “CCC” sequence and a signal of second “C” sequence from the right hand side in FIG.6 may be different.

If the signal values are differently measured, the present invention is such that the cantilever controller (170) may be utilized to move the polymerase (130) to another path (410) different from a reference path (400). That is, it is possible to perform a feedback control function using the cantilever controller (170) in response to the measured force of changed value, For example, as illustrated in FIG.7, the polymerase (130) attached to the tip (120) of the cantilever (120) is moved to contact a same type of dNTP (310) at a predetermined time and at a predetermined position. To this end, the AFM controller (160) can change the path of the moving means in response to the changed value of measured force.

Therefore, the present invention can analyze the sequence more accurately and quickly.

The previous description of the present invention is provided to enable any person skilled in the art to make or use the invention. Various modifications to the invention will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other variations without departing from the spirit or scope of the invention. Thus, the invention is not intended to limit the examples described herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

The present invention has an industrial applicability in that polymerase attached to a tip of a cantilever for AFM is used, and the polymerase is reacted with dNTP fixed to a substrate to increase a polymerization speed of target nucleic acid molecule, a polymerization reaction occurs at a predetermined height on the substrate to greatly increase accuracy of analysis due to no limit in read length, and a user can control a moving speed of a tip attached with the polymerase to adjust a speed of the polymerization reaction as desired, whereby the sequences can be analyzed more accurately.

Claims

An apparatus for determining sequences of nucleic acids using an AFM (Atomic Force Microscope), the apparatus characterized by: polymerase attached to a tip of a cantilever for AFM; a primer attachable to a single stranded target nucleic acid molecule polymerized by the polymerase; a substrate attached per type by dNTP hybridized with the single stranded target nucleic acid molecule; and moving means moving the polymerase on the substrate.
The apparatus of claim 1, further characterized by a dendron molecule attaching the polymerase to the tip of the cantilever.
The apparatus of claim 1, characterized in that the primer is a universal primer.
The apparatus of claim 1, characterized in that the dNTP is arranged in parallel on the substrate for each type.
The apparatus of claim 1, characterized in that the dNTP includes a plurality of dNTP sets formed with different types of dNTPs.
The apparatus of claim 1, characterized in that the dNTP is such that an outermost phosphate is fixed on the substrate.
The apparatus of claim 1, characterized in that the tip of the cantilever is charged with a positive polarity.
The apparatus of claim 1, characterized in that the moving means repetitively scan the dNTP per type.
The apparatus of claim 1, characterized in that the apparatus is further characterized by a controller measuring in real time changes in force generated from the tip of the cantilever by the polymerization.
The apparatus of claim 9, characterized in that the controller changes a path of the moving means in response to a changed value of the measured force.
A method for determining sequences of nucleic acids using an AFM (Atomic Force Microscope), the method characterized by: attaching polymerase to a tip of a cantilever for AFM; connecting a single strand of target nucleic acid molecule attached with a primer to the polymerase; and moving the polymerase connected with the single stranded target nucleic acid molecule on a substrate attached by dNTP per type.
The method of claim 11, characterized in that the step of attaching polymerase to a tip of a cantilever for AFM includes connecting dendron molecule to the tip of the cantilever, and attaching the polymerase to the dendron molecule.
The method of claim 11, characterized in that the step of connecting a single stranded target nucleic acid molecule to the polymerase includes; attaching a primer to the single stranded target nucleic acid molecule; and connecting the single stranded target nucleic acid molecule attached with the primer to the polymerase.
The method of claim 11, characterized in that the substrate is arranged in parallel with dNTP per type.
The method of claim 11, characterized in that the step of moving the polymerase on the substrate includes repetitively moving the polymerase on the substrate for each type of dNTP.
The method of claim 11, further characterized by applying a (+) bias to the tip of the cantilever.
The method of claim 11, further characterized by measuring in real time changes in force generated from the tip of the cantilever by polymerization of the single stranded target nucleic acid molecule and the dNTP.
The method of claim 17, further characterized by moving the polymerase to another path in response to the changed value of the measured force.