Apparatus suitable for single molecule sequencing and methods of sequencing nucleic acid molecules

Abstract

The present invention provides an apparatus suitable for single molecule sequencing and a method of sequencing a nucleic acid molecule. The apparatus includes at least one nano-well, a plurality of nucleic acid immobilization moieties, and a plurality of types of nucleic acid fragments. The nano-well has an observation zone. The nucleic acid immobilization moieties are disposed in or adjacent to the observation zone. The nucleic acid fragments are respectively immobilized to the nucleic acid immobilization moieties. At least one polymerase is disposed in the observation zone. A method of sequencing a nucleic acid molecule using the above apparatus is provided. The apparatus suitable for single molecule sequencing of the present invention provides the advantages of high throughput and short sample preparation time.

Description

[0001] [CROSS-REFERENCE TO RELATED APPLICATIONS]

[0002] This application claims priority to U.S. Provisional Application Serial No. 62 / 518,620, filed June 13, 2017. The entire disclosure of the above application is incorporated herein by reference and forms a part of the specification. TECHNICAL FIELD

[0003] The present invention relates to an apparatus and a method suitable for sequencing, and more particularly, to an apparatus suitable for single molecule sequencing and a method of sequencing a nucleic acid molecule. BACKGROUND

[0004] A conventional strategy for single molecule sequencing-by-synthesis (SBS) immobilizes one polymerase or one template within or near a reaction zone within a viewing space. One method in current single molecule sequencing methods includes the following steps. A nucleic acid template (e.g., DNA or RNA) to be sequenced is formed into a closed circle (circular template). Then, the nucleic acid template is captured by a polymerase immobilized within a detectable zone, where the polymerase has the properties of strand displacement activity and high processivity. The sequence of the template is then read several times via a single molecule sequencing-by-synthesis process. In this method, the final sequencing result is determined by a statistical consensus of the useful redundant reads. However, the method requires the polymerase to have strand displacement activity and high processivity, and the sequencing is terminated once the polymerase loses activity or releases the nucleic acid template. The method also requires a very precise loading process to maintain one template and have only one template in each detectable zone, so that those detectable zones without a template or with more than one template will be considered as failed sites. Therefore, such strict requirements narrow the operation window. SUMMARY

[0005] The present invention provides an apparatus suitable for single molecule sequencing with the advantages of high throughput and shortened sample preparation time.

[0006] The present invention provides a method of sequencing a nucleic acid molecule by using the above apparatus.

[0007] The present invention provides an apparatus suitable for single molecule sequencing. The apparatus includes at least one nano-well, a plurality of nucleic acid immobilization moieties, a plurality of types of nucleic acid fragments, and at least one polymerase. The nano-well has a viewing zone. The nucleic acid immobilization moieties are disposed in or adjacent to the viewing zone. The nucleic acid fragments are respectively immobilized to the nucleic acid immobilization moieties. The polymerase is disposed in the viewing zone.

[0008] In an embodiment of the present invention, the diameter of the observation area is between 10 nm and 500 nm, and the height of the observation area is less than 200 nm.

[0009] In embodiments of the present invention, the various types of nucleic acid fragments are linear nucleic acid fragments.

[0010] In embodiments of the present invention, the various types of nucleic acid fragments include 50 to 200 nucleotides in length.

[0011] In an embodiment of the present invention, the at least one polymerase is a single polymerase.

[0012] In an embodiment of the present invention, the nucleic acid fragment comprises more than 200 nucleotides in length.

[0013] In embodiments of the present invention, the at least one polymerase comprises a plurality of polymerases, and the device further comprises a plurality of polymerase immobilization portions. The polymerase immobilization portions are disposed in the observation area, and the polymerases are immobilized to the polymerase immobilization portions.

[0014] This invention provides a method for sequencing nucleic acid molecules, comprising the following steps: (a) providing at least one nanowell, wherein the nanowell has an observation region. (b) immobilizing multiple types of nucleic acid fragments into the observation region via multiple nucleic acid immobilization sites. (c) providing a primer and multiple labeled nucleotide analogs (also referred to as labeled nucleotides) into the nanowell, wherein the primer and one of the nucleic acid fragments form a primer-nucleic acid fragment complex in the observation region. (d) initiating a nascent strand synthesis reaction of the primer-nucleic acid fragment complex by using a polymerase and incorporating the labeled nucleotide analogs into the primer-nucleic acid fragment complex to form a nascent strand. (e) determining the sequence of the nucleic acid fragment by detecting the sequence of the incorporated labeled nucleotide analog.

[0015] In an embodiment of the present invention, the method further includes the following steps: (f) dissociating the newly formed chain; (g) repeating steps (c) to (f).

[0016] In an embodiment of the present invention, step (c) further includes adding the polymerase.

[0017] In an embodiment of the present invention, the method further includes the following step: immobilizing a plurality of polymerases onto the observation area via a plurality of polymerase immobilization portions, wherein the polymerase used in step (d) is one of the plurality of polymerases.

[0018] In an embodiment of the present invention, the step of fixing the plurality of polymerases is performed before step (c).

[0019] In an embodiment of the invention, the method further includes the following steps: (f) dissociating the nascent chains; (g) repeating steps (c) to (f), wherein the polymerase used in the repeated step (d) is one of the plurality of polymerases.

[0020] In embodiments of the present invention, the method for preparing the various types of nucleic acid fragments includes the following steps: Providing extracted genomic DNA. Dividing the extracted genomic DNA into multiple DNA fragments. Attaching one end of each of the multiple DNA fragments to multiple dsDNA (double-stranded DNA) adaptors, wherein the multiple dsDNA adaptors are immobilized on a solid phase. Attaching multiple labeled adaptors to the other ends of the multiple DNA fragments to form multiple labeled DNA fragments. Eluting multiple single-stranded DNA fragments by dissociating the multiple labeled DNA fragments.

[0021] In embodiments of the present invention, a washing process is further performed before engaging the plurality of labeled adapters and before eluting the plurality of single-stranded DNA fragments.

[0022] In embodiments of the present invention, the method for preparing the various types of nucleic acid fragments includes the following steps: Providing extracted genomic DNA. Splitting the extracted genomic DNA into multiple DNA fragments. Hybridizing a probe to one of the multiple DNA fragments. Extending the hybridized probe using a DNA polymerase and a labeled nucleotide analog to form a labeled nascent DNA fragment complex. Purifying the labeled nascent DNA fragment complex. Eluting multiple single-stranded DNA fragments by dissociating the labeled nascent DNA fragment complex. Attaching multiple adapters to one end of the single-stranded DNA fragments to form multiple labeled single-stranded DNA fragments.

[0023] In embodiments of the present invention, the preparation of the various types of nucleic acid fragments further includes performing a washing process in the step of purifying the labeled nascent DNA fragment complex.

[0024] Based on the above, the present invention provides an apparatus suitable for single-molecule sequencing and a method for sequencing nucleic acid molecules, wherein the apparatus includes at least one nanowell, multiple nucleic acid immobilization moieties, and various types of nucleic acid fragments. In this method, although multiple types of nucleic acid fragments are added simultaneously, only one primer-polymerase-nucleic acid complex is formed at any given time, and only one type of nucleic acid fragment (represented as a single molecule) is sequenced at any given time. In some embodiments, after sequencing one type of nucleic acid fragment, sequencing of the same type or another type of nucleic acid fragment is performed consecutively, eliminating the time required to add the nucleic acid fragment to be sequenced between these two sequencing processes. Therefore, the apparatus and method of the present invention have the advantages of high throughput and reduced sample preparation time.

[0025] To better understand the above content, several embodiments with accompanying diagrams are described in detail below. Attached Figure Description

[0026] The accompanying drawings are included to provide a further understanding of this disclosure, and are incorporated in and constitute a part of this specification. The drawings illustrate exemplary embodiments of this disclosure and, together with this description, serve to explain the principles of this disclosure;

[0027] Figures 1A to 1D This is a schematic diagram of a method for sequencing nucleic acid molecules according to an embodiment of the present invention;

[0028] Figure 2 A flowchart illustrating a method for sequencing nucleic acid molecules according to an embodiment of the present invention is shown;

[0029] Figure 3 This is a schematic diagram of a device suitable for single-molecule sequencing according to an embodiment of the present invention;

[0030] Figures 4A to 4G This is a schematic diagram of a method for preparing various types of nucleic acid fragments according to an embodiment of the present invention;

[0031] Figures 5A to 5G This is a schematic diagram of another method applicable to the preparation of various types of nucleic acid fragments according to an embodiment of the present invention. Detailed Implementation

[0032] The following detailed description is intended as an illustration of an exemplary device provided presently according to aspects of the invention, and is not intended to represent only the forms in which the invention can be made or utilized. Rather, it should be understood that the same or equivalent functions and components can be implemented through different embodiments, which are also intended to be encompassed within the scope and spirit of the invention.

[0033] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although any methods, apparatus, and materials similar to or equivalent to those described may be used to practice or test the invention, exemplary methods, apparatus, and materials are now described.

[0034] Figures 1A to 1D This is a schematic diagram of a method for sequencing nucleic acid molecules according to an embodiment of the present invention. Figure 2 A flowchart illustrating a method for sequencing nucleic acid molecules according to an embodiment of the present invention is shown. See also... Figure 1A and Figure 2 First, in step S100, at least one nanowell 110 is provided, wherein the nanowell 110 has an observation region 112. In some embodiments, an apparatus 100 including the nanowell 110 is provided, and the apparatus 100 is suitable for single-molecule sequencing. Specifically, the apparatus 100 includes the nanowell 110, a plurality of nucleic acid immobilization moieties 120a, 120b, various types of nucleic acid fragments 130a, 130b, and at least one polymerase 140a, 140b, 140c. In some embodiments, the apparatus 100 is suitable for sequencing nucleic acid fragments having more than 200 nucleotides in length. It should be noted that the number of nucleic acid immobilization moieties, nucleic acid fragments, and polymerases is illustrative for purposes of explanation and is not limited thereto. In other words, the number of nucleic acid immobilization moieties and nucleic acid fragments may be three or more, and the number of polymerases may be one or more. It should be noted that in some embodiments, polymerases 140a, 140b, 140c are already present in the apparatus 100; in some alternative embodiments, polymerases may be added subsequently.

[0035] The nanowell 110 has an observation region 112. In some embodiments, the observation region 112 is formed at or near the bottom of the nanowell 110. The bottom of the nanowell 110 may be transparent, and thus the sequencing process in the nanowell 110 can be observed or detected through the observation region 112. Therefore, the observation region 112 may be referred to as a detectable region, a sequencing site, or a single-molecule sequencing well. In some embodiments, the diameter of the observation region 112 is in the range of, for example, 10 nm to 500 nm. In some embodiments, the height of the observation region 112 is less than, for example, 200 nm. In some embodiments, the height of the observation region 112 is less than, for example, 100 nm. In some embodiments, the upper boundary of the observation region 112 may be curved.

[0036] Then, in step S110, multiple types of nucleic acid fragments 130a and 130b are immobilized onto the observation area 112 using multiple nucleic acid immobilization portions 120a and 120b. The nucleic acid immobilization portions 120a and 120b are located in or adjacent to the observation area 112. In some embodiments, nucleic acid immobilization portion 120a is located in the observation area 112, and nucleic acid immobilization portion 120b is located adjacent to the observation area 112. Nucleic acid fragments 130a and 130b are templates to be sequenced and are immobilized onto the nucleic acid immobilization portions 120a and 120b, respectively. Nucleic acid fragments 130a and 130b are different types of nucleic acid fragments, meaning that nucleic acid fragments 130a and 130b have different sequences. In some embodiments, the multiple types of nucleic acid fragments 130a and 130b are linear. In some embodiments, the nucleic acid fragments may be linear single-stranded or, for example, partially double-stranded with a hairpin structure. In some embodiments, nucleic acid fragments 130a and 130b include, for example, more than 200 nucleotides in length. In some alternative embodiments, the nucleic acid fragment has a fixing anchor at the 5' end or 3' end, preferably at the 3' end, and the nucleic acid fragment is fixed to the nucleic acid fixing portion by the fixing anchor. The fixing anchor may also be located at other locations (non-ends) of the nucleic acid fragment.

[0037] Nucleic acid immobilization portions 120a and 120b may be functional groups that immobilize nucleic acid fragments 130a and 130b via covalent bonding or affinity binding. Exemplary functional groups for covalent bonding may include, for example, thiols, primary amines, secondary amines, glycols, aldehydes, carboxyl groups, methacrylates, methacrylate silanes, aminosilanes, mercaptosilanes, aldehyde silanes, glycol silanes, azide silanes, cysteine, cysteine ​​derivatives, aminofunctional phosphates and their derivatives, epoxy, maleimide, silane maleimide, silane polyethylene glycol (PEG) maleimide, or silane PEG azide. Exemplary functional groups for affinity binding may include biotin, histidine, streptozotocin proteins, proteins, protein inhibitors, protein substrates, antibodies, antigens, or aptamers.

[0038] In some embodiments, polymerases 140a, 140b, and 140c are disposed in observation area 112. In some embodiments, nucleic acid fragments 130a and 130b have a longer length and are not completely disposed in observation area 112, and therefore, if polymerase 140d is not immobilized in observation area 112, the polymerase-nucleic acid complex may be disposed outside observation area 112. Therefore, device 100 also includes a plurality of polymerase immobilization portions 150a, 150b, and 150c located in observation area 112 for immobilizing polymerases 140a, 140b, and 140c. In this manner, the plurality of polymerases 140a, 140b, and 140c are located in observation area 112 by being immobilized to polymerase immobilization portions 150a, 150b, and 150c, respectively, and therefore the polymerase-nucleic acid complex is also located in observation area 112. It should be noted that the number of polymerase immobilization portions is illustrative for purposes of explanation and is not intended to limit the invention.

[0039] Then, refer to Figure 1B and Figure 2 In step S120, a primer 160a and a plurality of labeled nucleotide analogs 170 are provided to the at least one nanowell 110, wherein the primer 160a forms a primer-nucleic acid fragment complex 180a with one of the plurality of nucleic acid fragments 130a, 130b in the observation region 112. In some embodiments, only one primer 160a is added to the nanowell 110. In some embodiments, the primer 160a may have a sequence complementary to the nucleic acid fragment 130a, and thus the primer 160a is annealed to the nucleic acid fragment 130a to form the primer-nucleic acid fragment complex 180a in the observation region 112.

[0040] In some embodiments, nucleic acid fragments 130a and 130b comprise, for example, more than 200 nucleotides in length. In some embodiments, primer 160a may be annealed to the middle portion of nucleic acid fragment 130a so that the middle portion of nucleic acid fragment 130a serves as the starting point for the sequencing process. However, the invention is not limited thereto. In some alternative embodiments, the sequencing process may be initiated from the 5' end or anywhere else by using designed primers (probes) or by randomly priming nucleic acid fragments (not shown). In some embodiments, only one of nucleic acid fragments 130a and 130b may be primed at a time for sequencing by adding only one primer. In some embodiments, labeled nucleotide analogs 170 include, for example, labeled dATP, dCTP, dGTP, and dTTP. Labeled nucleotide analogs 170 may be fluorescently labeled nucleotide analogs.

[0041] Then, refer to Figure 1C and Figure 2In step S130, the synthesis of the nascent strand of primer-nucleic acid fragment complex 180a is initiated by using polymerases 140a, 140b, and 140c and incorporating the plurality of labeled nucleotide analogs 170 into the primer-nucleic acid fragment complex 180a. In some embodiments, one of polymerases 140a, 140b, and 140c (e.g., polymerase 140a) is used to synthesize the nascent strand 190a. In some embodiments, a single-molecule synthesis sequencing process is initiated after forming a primer-polymerase-nucleic acid complex containing one of nucleic acid fragments 130a and 130b, one of polymerases 140a, 140b, and 140c, and a primer 160a. That is, the sequencing process begins in the primer-polymerase-nucleic acid complex, and the sequencing process is initiated in the primer-polymerase-nucleic acid complex to synthesize the nascent strand 190a. It should be noted that although steps S110, S120 and S130 are described sequentially, these steps are performed simultaneously once the primers and nucleotide analogs are added, because the nucleic acid fragments and polymerases have already been added to the nanowells.

[0042] Then, in step S140, the sequence of nucleic acid fragment 130a is determined by detecting the sequence of the incorporated labeled nucleotide analog 170. In some embodiments, since only one labeled nucleotide analog 170 is incorporated into the primer-nucleic acid fragment complex 180a in the observation region 112 at any given time, the sequence of nucleic acid fragment 130a can be determined with high accuracy. In some embodiments, the labeled nucleotide analog 170 comprises labeled dATP, dCTP, dGTP, and dTTP with different fluorescence. In some embodiments, the sequence of nucleic acid fragment 130a can be determined by detecting the different fluorescence signals of the incorporated labeled dATP, dCTP, dGTP, and dTTP sequences.

[0043] Then, in step S150, the nascent strand 190a is dissociated. In some embodiments, the sequencing process performed by synthesizing the nascent strand 190a may be terminated due to the inactivation of polymerase 140a or the release of nucleic acid fragment 130a. However, after dissociating the nascent strand 190a, the sequencing process can be resumed by recapturing nearby polymerases 140a, 140b, and 140c. Specifically, if the sequencing process is terminated due to the inactivation of polymerase 140a, polymerase 140a may be removed as needed, and another nearby polymerase 140b or 140c may be recapturing to resume the sequencing process. If the sequencing process is terminated due to the release of nucleic acid fragment 130a, the same polymerase 140a or another nearby polymerase 140b or 140c may be recapturing to resume the sequencing process.

[0044] Then, refer to Figure 1D and Figure 2In step S160, steps S120 to S150 are repeated. In some embodiments, in the repeated step S120, a new primer 160b is added, and one of polymerases 140a, 140b, and 140c is recaptured to synthesize the nascent strand 190b. In some embodiments, polymerase 140b is used as an example, but this is for illustrative purposes only. In other words, as described above, in some alternative embodiments, polymerase 140a or another polymerase 140c may be recaptured in the repeated step S130 to synthesize the nascent strand 190a. Therefore, the sequencing process can be repeated several times by dissociating the nascent strand, providing a new primer, and recaptured one of the polymerases. In some embodiments, steps S120 to S150 are repeated to increase the sequence coverage of nucleic acid fragments 130a and 130b until the necessary accuracy of the common sequence is achieved. It should be noted that since polymerases 140a, 140b, and 140c are added to the nanowell from the outset, the addition of primers initiates the single-molecule synthesis sequencing process. It should also be noted that in some embodiments, although nucleic acid fragment 130b is fixed adjacent to observation region 112, the primer-polymerase-nucleic acid complex of nucleic acid fragment 130b, primer 160b, and polymerase 140b can also be formed and located within observation region 112. In other words, "adjacent" means that nucleic acid fragment 130b is close to observation region 112, and is separated from observation region 112 by a certain distance, wherein the distance is suitable for nucleic acid fragment 130b to form a primer-polymerase-nucleic acid complex within observation region 112. For single-stranded DNA molecules, the average length of each base is approximately 0.6 nm to 0.7 nm (the persistent length of single-stranded DNA and the length of each base are obtained by fluorescence correlation spectroscopy measurements using mean-field theory (Physica A: Statistical Mechanics and its Applications, Vol. 392, No. 5, March 1, 2013, pp. 1072-1079)). Although some portions of nucleic acid fragment 130b are not completely fixed within the observation region, a portion of the fragment is long enough to diffuse into the observation region and form a primer-polymerase-nucleic acid complex with the polymerase fixed within the observation region, thereby enabling sequencing. In some embodiments, the distance between the boundary of the observation region 112 and the nucleic acid fixation portion 120b of the nucleic acid fragment 130b must be within, for example, the range of L × 0.6 × 50% nm, in order to effectively form a primer-polymerase-nucleic acid complex in the observation region 112 between the boundary of the observation region 112 and the nucleic acid fixation portion 120b, where L is the fragment length in the bases.

[0045] In some embodiments, nucleic acid fragments 130a and 130b are immobilized in or adjacent to the same observation region 112, allowing for alternating and complete sequencing of the fragments within the same observation region 112 without cleaning and reloading another fragment. In other words, since nucleic acid fragments 130a and 130b are already present in the nanowells, after sequencing one type of nucleic acid fragment 130a, sequencing of the same type of nucleic acid fragment 130a or another type of nucleic acid fragment 130b can be performed consecutively, thus eliminating the time required to add the nucleic acid fragment to be sequenced between these two sequencing processes. In some embodiments, since nucleic acid fragments 130a and 130b are already present in the nanowells, after sequencing one nucleic acid fragment 130a, sequencing of the same nucleic acid fragment 130a or another nucleic acid fragment 130b can be performed consecutively, thus eliminating the time required to add the nucleic acid fragment to be sequenced between these two sequencing processes. It should be noted that although nucleic acid fragments 130a and 130b are simultaneously immobilized in or near the same observation region 112, only one nucleic acid fragment (represented as a single molecule) 130a is sequenced at any given time because only one primer-polymerase-nucleic acid complex is formed at any given time.

[0046] It should be noted that, although in Figures 1A to 1D In the present invention, nucleic acid fragments 130a and 130b comprise, for example, more than 200 nucleotides in length; however, the invention is not limited thereto. In some embodiments, such as Figure 3As shown, nucleic acid fragments 130c, 130d, and 130e comprise, for example, 50 to 200 nucleotides in length. In some embodiments, nucleic acid fragments 130c, 130d, and 130e are short fragments such as cell-free DNA or microRNA. In some embodiments, nucleic acid fragments 130c, 130d, and 130e are immobilized to nucleic acid immobilization portions 120c, 120d, and 120e, respectively. Because nucleic acid fragments 130c, 130d, and 130e have shorter lengths, they are substantially entirely disposed within or adjacent to observation region 112. Therefore, it is not necessary to immobilize polymerase 140d in observation region 112. Additionally, in some embodiments, adding a primer 160c and a polymerase 140d to nanowell 110 initiates the formation of the primer-polymerase-nucleic acid complex and performs single-molecule synthesis sequencing. In some embodiments, the sequencing process by synthesizing the nascent strand 190c may be terminated due to the inactivation of polymerase 140d or the release of nucleic acid fragment 130e. However, after dissociation of the nascent strand 190c, the sequencing process can be resumed by recapturing another newly added polymerase (not shown in the figure). Specifically, if the sequencing process is terminated due to the inactivation of polymerase 140d, polymerase 140d may be removed as needed, and the sequencing process can be resumed by recapturing a newly added polymerase (not shown in the figure). If the sequencing process is terminated due to the release of nucleic acid fragment 130e, the same polymerase 140d can be recapturing to resume the sequencing process. It should be noted that, although in Figure 3 The diagram schematically illustrates three nucleic acid fragments 130c, 130d, and 130e; however, the invention is not limited thereto. In some alternative embodiments, dozens or even hundreds of nucleic acid fragments may be loaded at once, and each fragment may be sequenced repeatedly. Thus, the sequencing capacity of device 100a can be multiplied.

[0047] Figure 3 This is a schematic diagram of a device suitable for single-molecule sequencing according to an embodiment of the present invention. The components of device 100a are similar to those of device 100, and the main difference between device 100 and device 100a is that device 100 is suitable for sequencing longer nucleic acid fragments (e.g., having more than 200 nucleotides in length), while device 100a is suitable for sequencing shorter nucleic acid fragments (e.g., having less than 200 nucleotides in length). The differences are described below. (Refer to...) Figure 3 The device 100a includes at least one nanowell 110 and multiple nucleic acid immobilization portions 120c, 120d, and 120e (for illustrative purposes, in...). Figure 3 The diagram schematically shows three fixed nucleic acid regions 120c, 120d, and 120e, and various types of nucleic acid fragments 130c, 130d, and 130e (for illustrative purposes, in...).Figure 3 The diagram schematically illustrates three types of nucleic acid fragments (130c, 130d, 130e) and a single polymerase 140d. Here, nucleic acid fragments 130c, 130d, and 130e comprise, for example, 50 to 200 nucleotides in length. Nucleic acid fragments 130c, 130d, and 130e are different types of nucleic acid fragments, meaning that they have different sequences. In some embodiments, polymerase 140d is disposed in observation area 112. In some embodiments, nucleic acid fragments 130c, 130d, and 130e have shorter lengths and are disposed entirely within or adjacent to observation area 112, and therefore the primer-polymerase-nucleic acid complex may be located in observation area 112. In some embodiments, the primer-polymerase-nucleic acid complex is a complex containing a primer, a polymerase, and a nucleic acid fragment. Therefore, polymerase 140d does not need to be separately immobilized in observation area 112.

[0048] In simple terms, in devices suitable for single-molecule sequencing, the multiple nucleic acid immobilization sites are located in or adjacent to the observation region of a nanowell, and the various types of nucleic acid fragments are immobilized to the nucleic acid immobilization sites. Although multiple types of nucleic acid fragments are added to the nanowell simultaneously, only one primer-polymerase-nucleic acid complex is formed at any given time, and only one type of nucleic acid fragment (represented as a single molecule) is sequenced at any given time. Furthermore, because the nucleic acid fragments are already present in the nanowell, sequencing of the same or another type of nucleic acid fragment can be performed consecutively after sequencing one type, thus eliminating the time required to add the nucleic acid fragment to be sequenced between these two sequencing processes. Therefore, the device and method of the present invention have the advantages of high throughput and reduced sample preparation time.

[0049] Figures 4A to 4G This is a schematic diagram of a method applicable to the preparation of various types of nucleic acid fragments in an embodiment of the present invention. Figures 5A to 5G This is a schematic diagram of another method applicable to the preparation of various types of nucleic acid fragments in another embodiment of the present invention.

[0050] Reference Figures 4A to 4GA method suitable for preparing the various types of nucleic acid fragments includes the following steps. First, extracted genomic DNA 210 is provided. Then, the extracted genomic DNA 210 is split into a plurality of DNA fragments 212. Then, one end 212a of the DNA fragments 212 is attached to a dsDNA adaptor 220, wherein the dsDNA adaptor 220 is immobilized on a solid phase 230. Then, a washing process is performed to wash away unbound DNA fragments 212. Then, a labeled adaptor 240 is attached to the other end 212b of the DNA fragments 212 attached to the solid phase 230 to form a labeled DNA fragment 214. In some embodiments, the labeled adaptor 240 is, for example, a partially double-stranded DNA having a single-stranded portion, wherein a label F is labeled on the end of the single-stranded portion. In some embodiments, the label F is, for example, biotin. Then, a washing process is performed to wash away unattached labeled adaptor 240. Then, the plurality of single-stranded DNA fragments 214a are eluted by dissociating the labeled DNA fragments 214. Here, single-stranded DNA fragment 214a can be directly used as a nucleic acid fragment for sequencing.

[0051] Reference Figures 5A to 5G Another method for preparing the various types of nucleic acid fragments includes the following steps. First, extracted genomic DNA 310 is provided. Then, the extracted genomic DNA 310 is split into multiple DNA fragments 312. Then, a probe 320 is hybridized to one of the DNA fragments 312. Then, the hybridized probe 322 is extended using DNA polymerase 330 and a labeled nucleotide analog 340 to form a labeled nascent DNA fragment complex 313. In some embodiments, each of the labeled nucleotide analogs 340 contains a label, and the label is, for example, biotin. Then, the labeled nascent DNA fragment complex 313 is purified. A washing process is performed to wash away unlabeled DNA fragments 312. In some embodiments, the labeled nascent DNA fragment complex 313 is purified using magnetic beads 350 coated with streptomycin protein and a magnet 360. Then, multiple single-stranded DNA fragments 313a are eluted by dissociating the labeled nascent DNA fragment complex 313. Then, anchor 370 is attached to one end 313a1 (or the other end 313a2) of the single-stranded DNA fragment 313a to form a labeled single-stranded DNA fragment 314. Here, the labeled single-stranded DNA fragment 314 can be directly used as a nucleic acid fragment for sequencing.

[0052] In some embodiments, each sequencing process may yield a partial sequence of a nucleic acid fragment. The partial sequences may begin at the same initiation point or at different initiation points and end at different endpoints. The sequencing process may be repeated several times to obtain several partial sequences within a single nanowell. Then, by aligning the several partial sequences together, a longer common sequence of the nucleic acid fragment can be obtained.

[0053] In summary, this invention provides an apparatus suitable for single-molecule sequencing and a method for sequencing nucleic acid molecules. The apparatus includes at least one nanowell, multiple nucleic acid immobilization regions, and various types of nucleic acid fragments. In some embodiments, the sequencing process can be initiated by adding a primer or a polymerase, thus ensuring that only one primer-polymerase-nucleic acid complex is formed. Therefore, at any given time, only one labeled nucleotide analog is incorporated into the primer-polymerase-nucleic acid complex, and this labeled nucleotide analog can be detected as a sequencing result through the observation region of the nanowell. Thus, sequencing results can be obtained accurately without interference. Furthermore, because the nucleic acid fragment is added to the nanowell from the beginning, nucleic acid fragments can be sequenced continuously, eliminating the time required to add the nucleic acid fragments to be sequenced. Moreover, since multiple nucleic acid fragments and / or multiple polymerases are loaded into one nanowell during a single loading operation, loading failure is prevented. Therefore, the apparatus and method of this invention have the advantages of high throughput and reduced sample preparation time.

[0054] It will be apparent to those skilled in the art that various modifications and variations can be made to the disclosed embodiments without departing from the scope or spirit of this disclosure. In view of the foregoing, this disclosure is intended to cover various modifications and variations, provided that such modifications and variations fall within the scope of the foregoing claims and their equivalents.

Claims

1. A device suitable for single-molecule sequencing, characterized in that, include: At least one nanowell, wherein the at least one nanowell comprises: Observation area; Multiple nucleic acid fixation sites are disposed in or near the observation area; Multiple types of nucleic acid fragments are respectively immobilized to the plurality of nucleic acid immobilization regions; A single primer; and At least one polymerase is set in the observation area.

2. The device according to claim 1, characterized in that, The diameter of the observation area is between 10 nm and 500 nm, and the height of the observation area is less than 200 nm.

3. The device according to claim 1, characterized in that, The various types of nucleic acid fragments mentioned are linear nucleic acid fragments.

4. The device according to claim 1, characterized in that, The various types of nucleic acid fragments range in length from 50 to 200 nucleotides.

5. The device according to claim 4, characterized in that, The at least one polymerase is a single polymerase.

6. The device according to claim 1, characterized in that, The various types of nucleic acid fragments comprise more than 200 nucleotides in length.

7. The device according to claim 6, characterized in that, The at least one polymerase includes a plurality of polymerases, and the device further includes a plurality of polymerase immobilization portions located in the observation area, wherein the plurality of polymerases are immobilized to the plurality of polymerase immobilization portions.

8. A method for sequencing nucleic acid molecules, characterized in that, include: (a) Provide at least one nanowell, wherein the at least one nanowell includes an observation region and a plurality of nucleic acid immobilization portions, and the plurality of nucleic acid immobilization portions are disposed in or adjacent to the observation region; (b) Adding multiple types of nucleic acid fragments to the observation region of the at least one nanowell, such that the multiple types of nucleic acid fragments are immobilized to the observation region of the at least one nanowell through the multiple nucleic acid immobilization portions; (c) Providing a single primer and a plurality of labeled nucleotides into the at least one nanowell, wherein the primer forms a primer-nucleic acid fragment complex with one of the plurality of nucleic acid fragments in the observation region; as well as (d) The nascent chain synthesis reaction of the primer-nucleic acid fragment complex is initiated by using a polymerase and incorporating the plurality of labeled nucleotides into the primer-nucleic acid fragment complex to form a nascent chain; as well as (e) The sequences of the various types of nucleic acid fragments are determined by detecting the sequences of the multiple labeled nucleotides after inclusion.

9. The method according to claim 8, characterized in that, It also includes the following steps: (f) Dissociate the newly formed chain; and (g) Repeat steps (c) to (f).

10. The method according to claim 8, characterized in that, The diameter of the observation area is between 10 nm and 500 nm, and the height of the observation area is less than 200 nm.

11. The method according to claim 8, characterized in that, The various types of nucleic acid fragments range in length from 50 to 200 nucleotides.

12. The method according to claim 11, characterized in that, Step (c) also includes adding the polymerase.

13. The method according to claim 8, characterized in that, The various types of nucleic acid fragments comprise more than 200 nucleotides in length.

14. The method according to claim 13, characterized in that, It also includes immobilizing a plurality of polymerases onto the observation area via a plurality of polymerase immobilization portions, wherein the polymerase used in step (d) is one of the plurality of polymerases.

15. The method according to claim 14, characterized in that, The step of fixing the plurality of polymerases is performed before step (c).

16. The method according to claim 14, characterized in that, It also includes the following steps: (f) Dissociate the newly formed chain; and (g) Repeat steps (c) to (f), wherein the polymerase used in the repeated step (d) is one of the plurality of polymerases.

17. The method according to claim 8, characterized in that, The methods for preparing the various types of nucleic acid fragments include: Provide extracted genomic DNA; The extracted genomic DNA was split into multiple DNA fragments; One end of the plurality of DNA fragments is attached to a plurality of double-stranded DNA linkers, wherein the plurality of double-stranded DNA linkers are immobilized on a solid phase. Multiple labeled adaptors are attached to the other end of the multiple DNA fragments to form multiple labeled DNA fragments; and Multiple single-stranded DNA fragments are eluted by dissociating the multiple labeled DNA fragments.

18. The method according to claim 17, characterized in that, The process further includes performing washing processes before engaging the plurality of labeled adapters and before eluting the plurality of single-stranded DNA fragments.

19. The method according to claim 8, characterized in that, The methods for preparing the various types of nucleic acid fragments include: Provide extracted genomic DNA; The extracted genomic DNA was split into multiple DNA fragments; The probe is hybridized to one of the plurality of DNA fragments; The hybridized probe was extended using DNA polymerase and labeled nucleotides to form a labeled nascent DNA fragment complex; The labeled nascent DNA fragment complex was purified; Multiple single-stranded DNA fragments were eluted by dissociating the labeled nascent DNA fragment complex; and Multiple anchors are attached to one end of the multiple single-stranded DNA fragments to form multiple labeled single-stranded DNA fragments.

20. The method according to claim 19, characterized in that, Also includes: A washing process is performed in the step of purifying the labeled nascent DNA fragment complex.

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