Apparatus for extracting strands of nucleic acids from biological samples

Abstract

An apparatus for extracting strands of nucleic acids from biological samples, the apparatus comprising: a sample container for holding a biological sample in use; at least one rotatable magnetic element arranged to be positioned in the sample container and comprising a surface for binding nucleic acids; and a magnetic field source, wherein the apparatus is arranged such that, in use, the magnetic field source causes the at least one rotatable element to rotate and wind at least one strand of nucleic acid thereon.

Description

[0001] Apparatus for extracting strands of nucleic acids from biological samples

[0002] TECHNICAL FIELD

[0003] The present invention relates to a method and apparatus for extraction of nucleic acids, in particular, for high molecular weight DNA.

[0004] BACKGROUND

[0005] Many modern fields, such as genetics and disease research, rely on data obtained from tools such as DNA sequencing to enable researchers to investigate gene functions and provides a better understanding of complex genome structure and function.

[0006] However, these tools ultimately rely on the obtaining of high-quality nucleic acid and DNA samples which can be an onerous and complicated process, often performed manually.

[0007] There are several known techniques for such extraction. However, these have several problems and do not meet all the requirements described above.

[0008] For example, traditionally, it is possible to extract DNA from a biological sample by using a phenol, chloroform and isoamyl alcohol mixture followed by ethanol precipitation. However, this method can be time consuming involving several manual steps.

[0009] More recent approaches involve placing two glass beads in a sample container with the biological sample. The sample container is then vertically rotated to create a rolling motion, enabling DNA to wind around the glass beads for collection. The glass beads may have a smooth surface aiding in the binding and eluting of the DNA. The beads may be rotated manually by hand, or by using a benchtop device. However, this limits the number of samples that can be processed per batch, the ability to automate some or any of the steps of the assay, and may cause longer DNA molecules to shear. There accordingly exists a need for a DNA extraction method and apparatus capable of automating DNA extraction in a manner that is easy and efficient and is comparable in terms of quality and output to existing approaches.

[0010] SUMMARY OF THE INVENTION

[0011] The present invention seeks to provide a method and apparatus for nucleic acid extraction, in particular, for high molecular weight DNA.

[0012] According to a first aspect of the invention, there is provided an apparatus for extracting strands of nucleic acids from biological samples, the apparatus comprising: a sample container for holding a biological sample in use; at least one rotatable permanently magnetic element arranged to be positioned in the sample container and comprising a surface for binding nucleic acids; and a magnetic field source, wherein the apparatus is arranged such that, in use, the magnetic field source causes the at least one rotatable element to rotate and wind at least one strand of nucleic acid thereon.

[0013] By rotating at least one rotatable element using a magnetic field, it is possible to wind a strand of nucleic acid in a controllable and predictable manner. Furthermore, it allows rotation of the rotatable element without requiring rotation of the sample container. This advantageously avoids the need to place the sample container in a benchtop vertical mixer, or the like, in order to wind nucleic acids. This substantially improves automation capabilities, since the sample container does not need to be moved between different processing stations. The sample container may form part of a well plate. Further by using one or more permanent magnetic elements on to which to wind the nucleic acid ensures greater control of the winding process and therefore greater control of the characteristics of the collected sample.

[0014] Moreover, the magnetic properties of the rotatable element can be exploited to firstly achieve winding of nucleic acid, and secondly to magnetically manipulate the rotatable element, and therefore the bound nucleic acid, using a static magnetic field. This manipulation can be used when performing subsequent method steps, such as washing and elution, allowing more control over sample handling and further preventing loss of or damage to the nucleic acid material.

[0015] The sample container is further configured to hold a solution containing the biological sample.

[0016] Optionally, at least one of the rotatable element is spherical.

[0017] Optionally, the diameter of the rotatable element is greater than 0.1 mm. Such a diameter is favourable for extracting a long strand of nucleic acids.

[0018] Optionally, at least one of the rotatable element is dog bone-shaped or caltropshaped.

[0019] Optionally, there are two rotatable elements manipulated by a magnetic field source to generate a stepping motion. Optionally, the rotatable element comprises glass.

[0020] Optionally, the rotatable element has a coated surface, and the rotatable element may be coated with Ni-Cu-Ni or Silica.

[0021] Optionally, the magnetic field source comprises a plurality of permanent magnets, or a magnetic array.

[0022] Optionally, the magnetic field source creates a rotating magnetic field.

[0023] Optionally, the magnetic field source is arranged to rotate the rotatable element to rotate at a speed in the range of substantially 10 RPM to 500 RPM. The system may have a controller capable of controlling the magnetic field to control the speed of rotation so as to adjust the characteristics of the collected samples. The controller may also be arranged to control the magnetic field to create a stepped motion in the rotatable elements to further control the sample collection process according to sample generation requirements. Optionally, the apparatus comprises a plurality of wells defining a plurality of biological sample containers, each having at least one rotatable element positioned therein and arranged to contain a biological sample in use.

[0024] Optionally, the plurality of wells has between 2 and 384 biological sample containers.

[0025] Optionally, the magnetic field source is arranged to rotate each respective rotatable element in each of the plurality of wells.

[0026] Optionally, the sample container is a consumable.

[0027] A kit and method for performing the invention is also provided.

[0028] According to a further aspect of the invention there is provided a method of extracting at least one strand of nucleic acid from a biological sample held in a solution in a sample container comprising the steps of:

[0029] (a) applying a magnetic field to at least one permanent magnetic rotatable element in the solution to rotate and wind at least one strand of nucleic acid thereon; and

[0030] (b) magnetically manipulating the at least one magnetic rotatable element in a preceding or subsequent sample preparation step to thereby extract the at least one strand.

[0031] Optionally, the magnetic manipulation of the at least one magnetic rotatable element is performed using a permanent magnet, an electromagnet or a ferromagnetic material.

[0032] Optionally, step (b) comprises applying, prior to step (a), a changing magnetic field to at least one magnetic rotatable element to mix the solution during lysis of the biological sample.

[0033] Optionally, step (b) comprises holding the at least one magnetic rotatable element in a position in order to allow extraction, addition and / or mixing of solution. Optionally, step (b) comprises moving, after step (a), the at least one magnetic rotatable element from the sample container to at least one further container.

[0034] Optionally, step (b) comprises applying, after step (a), a magnetic field to rotate the at least one magnetic rotatable element in order to re-suspend the nucleic acid in a solution.

[0035] As will be appreciated from the above, the present invention enables nucleic acid to be extracted from a biological sample for further processing, such as sequencing library preparation. The present invention prevents nucleic acid shearing during extraction by gentle agitation and binding, in a minimally destructive manner, resulting in high molecular weight DNA. The present invention may be automated and scalable which reduces labour cost, and improves throughput and reliability (i.e. , for long read sequencing applications).

[0036] BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Embodiments of the invention will now be described, by way of example only, with reference to the accompanying drawings, in which:

[0038] Figure 1 is an illustration of an existing apparatus for winding DNA;

[0039] Figure 2 illustrates how a rotatable magnetic element may be manipulated by a single external bar magnet according to an embodiment of the invention;

[0040] Figure 3 is a bar magnet rotatable along its horizontal axis that may be used in the invention;

[0041] Figure 4 illustrates a method of performing the present invention by using an existing process;

[0042] Figure 5 illustrates how the present invention can improve existing processes for DNA extraction; Figure 6 illustrates that there is an optimal bead size to volume ratio employed in the invention, as well as the ability to employ the invention at different reaction scales;

[0043] Figures 7a and 7b are figures showing gel electrophoresis results for two methods employing the invention;

[0044] Figure 8 is a graph showing HMWgDNA module binding time trial results from a manual process and employing the system and method of the invention;

[0045] Figure 9 is a graph showing the relationship between rotation speed of an element or elements and DNA shearing; and

[0046] Figure 10 is a table showing the characteristics of HMWgDNA samples produced by the invention.

[0047] DETAILED DESCRIPTION

[0048] The following further describes specific embodiments of this application in detail with reference to accompanying drawings.

[0049] Figure 1 illustrates an existing apparatus for winding DNA. In the illustrated example, glass beads 100 can be used to create a rolling motion enabling DNA to wind around. The rotating motion is usually performed manually by hand, or with the aid of a benchtop instrument 101. The stepping action of the beads when inverted is key to the gentle binding of long DNA strands 102.

[0050] Figure 2 illustrates an apparatus for extracting strands of nucleic acids from biological samples according to an embodiment of the present invention. As shown in figure 2, at least one rotatable permanently magnetic element 200 (in this example two elements 200 are shown) and a biological sample 201 are placed in a sample container 203. An external magnetic element 204 is placed adjacent to the container 203, in this case beneath it. In operation the magnetic pole of the magnetic element 204 is switched such that, in use, the change in magnetic field causes the at least one rotatable element 200 to rotate and wind nucleic acids or DNA contained in the sample on to the at least one rotatable element 200. Preferably, the rotatable magnetic element comprises at least two magnetic beads 200. Each magnetic bead may be at least 0.1 mm in diameter. In some embodiments, the magnetic beads may be at least 0.5 mm in diameter, and can be up to 100 mm in diameter.

[0051] A preferred embodiment of the invention is the provision two spherical permanent magnets as the magnetic elements 200 attached magnetically so that their magnetic field is aligned, generating a larger magnetic field. When the combined elements are manipulated by the external magnetic element 204 they may rotate around an oscillating centre of rotation, allowing movement in a stepping motion, replicating the benchtop process of figure 1 and enabling efficient nucleic acid binding. It should be noted that the configuration of the external magnetic element 204 can change depending on system architecture. For example, a rotating magnetic source can be used as the external element 204 to cause the two magnetic elements 200 to step over each other, mimicking the benchtop protocol. By mimicking the benchtop protocol, the invention has the added advantage of enabling scalable automation.

[0052] The rotatable elements 200 are provided by permanent magnets, the movement of which plays a role in the capture of nucleic acids, unlike ferromagnetic or paramagnetic alternatives such as SPRI beads as have been used in the prior art.

[0053] In some embodiments, the rotatable magnetic element(s) 200 may comprise two or more spherical permanent magnets, attached magnetically to define an elongate shape. In other embodiments, the rotatable magnetic element(s) 200 can be single dog-bone shapes, akin to a single rounded cylinder. In an alternative embodiment, the rotatable magnetic element(s) 200 can be caltrop-shaped. Having a caltrop-shaped rotatable magnetic element 200 will also produce a semi- chaotic walking motion thereby aiding the mixing and winding of the nucleic acid or DNA from the biological sample.

[0054] The rotatable magnetic element(s) 200 may be coated with a Ni-Cu-Ni coating to prevent oxidation and may be between 0.1 mm to 100 mm in diameter, depending on the volume of sample container and the biological sample being processed. In other embodiments, the rotatable magnetic element(s) 200 may be coated with a smooth silica surface.

[0055] The rotatable magnetic element(s) 200 may be manipulated by a single external magnet 204. The external magnetic element 204 generates a rotating or oscillating magnetic field which causes the rotatable magnetic element(s) 200 to correspondingly rotate at the same rate about an oscillating horizontal axis. The oscillation of the horizontal axis preferably has at least a vertical component. The rotation motion gently mixes the fluid, and ‘winds up’ the nucleic acid or DNA around the surface of the rotatable magnetic element(s) 200. The size of the nucleic acid fragments may be controlled by the speed of the rotation motion. For example, a faster speed (i.e., 500 RPM or higher) is likely to result in smaller nucleic acid fragments. Conversely, a lower speed (i.e., around 10 RPM) is likely to result in larger nucleic acid fragments. This is shown in figure 9, where it can be seen that increased rotational speed induces DNA shearing, and through use of this control of the process an extraction process that avoids the need for needle sheering can be employed, giving it an advantage over other techniques.

[0056] In some embodiments, as shown in figure 3, the external magnetic element 204 may be a square rod rotatable along its horizontal axis and is magnetised across one of its short sides 300. It would be reasonably understood though that any kind of magnet or magnetic field source may be utilised to achieve the intended magnetic field. This includes, for example, a bar magnet, electromagnet, or multiple sources such as in a magnetic array.

[0057] In some embodiments, the present invention may comprise a plurality of wells. Each well represents a biological sample container 203 as described above, each having at least one rotatable element 200 positioned therein. The plurality of wells can contain between 2 wells for larger volume extraction, up to 384 wells for small volume extraction. The plurality of wells can be a provided as a single use consumable. For example, the plurality of wells may comprise disposable test tubes positioned on a holding rack. Alternatively, the plurality of wells may be integral with the holding rack. In this scenario, it would be reasonably understood that either the plurality of wells, the holding rack, or both can be a consumable. This also means that a single consumable may be used to complete an entire automated assay. Indeed, it will be appreciated that one of the benefits of the invention is that by employing the process in a container with the rotating element(s) it is possible for a number of processing steps to be carried out within that same container with the purified sample only being removed at the end. This in turn provides the capability for a higher density of extraction as well as improved automation.

[0058] A single external magnetic source 204, such as the one shown in figure 3, can be utilised to actuate at least two rows of wells. The rotation axis of the magnet can be vertical or horizonal. For example, a pure vertical axis with no vertical mixing motion may be more suitable for low liquid volumes or, when a larger magnet is used, where vertical mixing is not required. Conversely, with a predominately horizontal axis of rotation, the rotatable magnetic elements 200 ‘walk’ over each other providing vertical mixing and higher fluid displacement. The winding motion spools the nucleic acids together enabling the rotatable magnetic elements to wind nucleic acid effectively and efficiently.

[0059] In practice, the present invention can be used as a complement to existing reagents for extracting nucleic acid, and commercially available kits. The existing reagents include, for example, lysis reagents comprising salt, enzymes and / or detergents. The nucleic acid may be precipitated using Isopropyl alcohol (I PA) and ethanol, with elution of the nucleic acid in water or TE buffer. The present invention is therefore compatible with many existing processes for nucleic acid extraction.

[0060] The present invention can be implemented using the process illustrated in figure 4. Cells are first lysed on the bench 400 before being pipetted into a well plate or test tube containing a bead 401. A magnet is then used to rotate the bead 402. Isopropyl alcohol (I PA) is then added to precipitate nucleic acid 403. The sample is then rotated for at least 1 min, at room temperature 404 before the supernatant is removed with a pipette 405, leaving behind nucleic acid bound to the beads. The sample can be rotated for any period as necessary (i.e. , 8 mins). The bound nucleic acid is then washed twice 406 before being eluted at 56 degrees Celsius for 10 minutes at 300 rpm of agitation 407.

[0061] Whilst this process can result in the extraction of high-quality nucleic acid samples, there is a risk that fluid exchange steps and sample handling can result in shearing or loss of the nucleic acid. The present invention allows these steps to be replaced or changed to protect the nucleic acid, as illustrated in figure 5. As with the previous example, cells may be lysed on beads 500 or lysed on the bench 501 before being pipetted into a well plate containing a bead 502 and rotated with a magnetic field 503. I PA is added to the biological sample to precipitate DNA 504 and rotated for at least 1 min. The sample can be rotated for any period as necessary (i.e., 8 mins) 505.

[0062] After step 505, the two different ways of magnetically manipulating the beads can be performed, using a permanent or electro-magnet (which may be the same as or different to the magnetic field source used for rotation of the beads) or using a ferromagnetic material outside the sample container.

[0063] In a first, the magnetic beads may be moved into a new reagent well for a first washing step, and then into a second reagent well for a second washing step 509. The magnetic beads are then either moved into an elution well, rotated, and heated 510, or agitated and heated on or off rig 509.

[0064] Alternatively, the beads may be magnetically moved to the side of the well, allowing easy removal and addition of reagents 506, going through two wash cycles 507, and then either heated and mixed with magnetic rotation 510, or agitated and heated on or off rig 509.

[0065] As the bead may be moved magnetically, the sample is easily accessible as the bead can be moved to the side of the well.

[0066] As explained above, the magnetic beads may be at least 0.1 mm in diameter. In some embodiments, the magnetic beads may be at least 0.5 mm in diameter and can be up to 100 mm in diameter. In this respect, as shown in figure 6, the present invention can be modified and adapted for optimal nucleic acid extraction by adjusting the bead size (and composition) to volume ratio. Larger beads may be used in larger volumes 600, smaller beads in smaller volumes 602, or an optimal balance in between 601 .

[0067] Figures 7a and 7b show gel electrophoresis results from two methods performed using the system of the invention. In figure 7a there are shown gel electrophoresis results of purified gDNA. This shows the invention can purify 60kb+ fragments of DNA starting from a complex lysate. In figure 7b there are shown gel electrophoresis results of purified gDNA where the invention starts with cells as an input. This highlights that the invention can be used process from cells to purified HMW gDNA in a single pot.

[0068] Figure 8 is a graph showing log-transformed DNA sequencing read length for HMWg DNA samples produced firstly using two standard manual processes, followed by six samples produced using the invention, from which it can be seen that the results from the invention are comparable to those produced manually.

[0069] Figure 9 has been discussed above, it shows how the rotational speed of rotating magnetic elements 200 in the invention can affect the sheering of the samples produced. This feature of the invention can be used to control the nature of the sample outputs by controlling the rotation, thus allowing a user to focus the operation of the system to a particular sample output characteristic without manual intervention.

[0070] Figure 10 is a table showing the characteristics of HMWgDNA samples produced by the invention, indicating the level of quality and purity which avoids the need for further processing of the samples in down stream steps in any analysis in which the samples are used.

[0071] It is understood that the described embodiments and figures in this application are merely used as illustrative examples and are not used to limit the scope of embodiments of this application. Any feature described in relation to any one embodiment may be used alone, or in combination with other features described, and may also be used in combination with one or more features of any other of the embodiments, or any combination of any other of the embodiments. Many modifications will be apparent to those skilled in the art without departing from the scope of the present invention. Equivalents and modifications not described above may also be employed without departing from the scope of the invention, which is defined in the accompanying claims, and as hereinbefore described with reference to the accompanying drawings.

Claims

CLAIMS1. An apparatus for extracting strands of nucleic acids from biological samples, the apparatus comprising: a sample container for holding a biological sample in use; at least one rotatable permanent magnetic element arranged to be positioned in the sample container and comprising a surface for binding nucleic acids; and a magnetic field source, wherein the apparatus is arranged such that, in use, the magnetic field source causes the at least one rotatable element to rotate and wind at least one strand of nucleic acid thereon.

2. The apparatus of claim 1 , wherein at least one of the rotatable element is spherical.

3. The apparatus of claim 2 wherein the diameter of the rotatable element is greater than 0.1 mm.

4. The apparatus of claim 1 , wherein at least one of the rotatable element is dog bone-shaped or caltrop-shaped.

5. The apparatus of any preceding claim, wherein the rotatable element is configured to rotate around an oscillating axis of rotation.

6. The apparatus of any one of any one of the preceding claims, wherein the rotatable element comprises glass and / or is coated with Ni-Cu-Ni or Silica.

7. The apparatus of any one of claims 1 to 6, wherein the magnetic field source comprises a plurality of permanent magnets, or a magnetic array.

8. The apparatus of claim 7, wherein the magnetic field source is arranged to create a rotating magnetic field.

9. The apparatus of any one of claims 1 to 8, wherein the magnetic field source is arranged to rotate the rotatable element to rotate at a speed in the range of substantially 10 RPM to 500 RPM.

10. The apparatus of any one of claims 1 to 9, comprising a plurality of wells defining a plurality of biological sample containers, each having at least one rotatable element positioned therein and arranged to contain a biological sample in use.

11. The apparatus of claim 10, wherein the plurality of wells has between 2 and 384 biological sample containers.

12. The apparatus of claim 10 or 11 wherein the magnetic field source is arranged to rotate each respective rotatable element in each of the plurality of wells.

13. The apparatus of claims 1 to 12, wherein the sample container is a single use consumable.

14. A method of extracting at least one strand of nucleic acid from a biological sample held in a solution in a sample container comprising the steps of:(a) applying a magnetic field to at least one permanent magnetic rotatable element in the solution to rotate and wind at least one strand of nucleic acid thereon; and(b) magnetically manipulating the at least one magnetic rotatable element in a preceding or subsequent sample preparation step to thereby extract the at least one strand.

15. The method of claim 14, wherein the magnetic manipulation of the at least one magnetic rotatable element is performed using a permanent magnet, an electromagnet or a ferromagnetic material.

16. The method of claim 14 or claim 15, wherein step (b) comprises applying, prior to step (a), a changing magnetic field to at least one magnetic rotatable element to mix the solution during lysis of the biological sample.

17. The method of any of claims 14 to 16, wherein step (b) further comprises holding the at least one magnetic rotatable element in a position in order to allow extraction, addition and / or mixing of solution.

18. The method of any of claims 14 to 17, wherein step (b) further comprises moving, after step (a), the at least one magnetic rotatable element from the sample container to at least one further container.

19. The method of any of claims 14 to 18, wherein step (b) further comprises applying, after step (a), a magnetic field to rotate the at least one magnetic rotatable element in order to re-suspend the nucleic acid in a solution.

Images