Patent Application
20190203273
This invention relates to a method and associated biological probe systems for detecting and characterising single nucleotides. It is especially suitable for use in the sequencing of DNA or RNA.
Next generation sequencing of genetic material is already making a significant impact on the biological sciences in general and medicine in particular as the unit cost of sequencing falls in line with the coming to market of faster and faster sequencing machines.
In our previous applications WO 2014/053853, WO 2014/053854, WO2014/167323, WO2014/167324 and WO2014/111723 we have described a new sequencing method which involves progressive digestion of a polynucleotide analyte to generate an ordered stream of single nucleotides, preferably a stream of single nucleoside triphosphates, each of which can be captured one-by-one into corresponding droplets in a microdroplet stream. Thereafter, each droplet can be chemically and/or enzymatically manipulated to reveal the particular single nucleotide it originally contained. In one embodiment, these chemical and/or enzymatic manipulations comprise a method involving the use of one or more two-component oligonucleotide probe types each of which is adapted to be able to selectively capture one of the single nucleotide types from which the analyte is constituted. Typically, in each of such probe types, one of the two oligonucleotide components comprises characteristic fluorophores and in the probe's unused state the ability of these fluorophores to fluoresce remains extinguished by virtue of the presence of quenchers located close-by or by self-quenching. In use, when the probe has captured its corresponding single nucleotide, it is rendered susceptible to subsequent exonucleolysis thereby liberating the fluorophores from the quenchers and/or each other enabling them to fluoresce freely. By this means, the original single nucleotide present in each droplet can be inferred indirectly by spectroscopic means.
Variants of this method have been described in other of our pending applications including WO201405385 and WO2016012789; the latter involving the use of a three-component probe system. In particular, WO2016012789 describes an improved method characterised by the steps of (1) generating a stream of single nucleoside triphosphates by progressive digestion of the nucleic acid; (2) producing at least one substantially double-stranded oligonucleotide used probe by reacting in the presence of a polymerase and a ligase at least one of the single nucleoside triphosphates with a corresponding probe system comprising (a) a first single-stranded oligonucleotide labelled with e.g. characteristic fluorophores in an undetectable state and (b) second and third single-stranded oligonucleotides capable of hybridising to complementary regions on the first oligonucleotide; (3) digesting the used probe with an enzyme having double-stranded exonucleolytic activity to yield the fluorophores in a detectable state and a single-stranded fourth oligonucleotide which is at least in part the sequence complement of the first oligonucleotide; (4) reacting the fourth oligonucleotide with another first oligonucleotide to produce a substantially double-stranded oligonucleotide product corresponding to the used probe; (5) repeating steps (3) and (4) in a cycle and (6) detecting the fluorophores released in each iteration of step (3). This method has the advantage that by iterating steps (3) and (4) in a cycle the fluorescence signal can be made to grow strongly thereby improving the overall sensitivity and therefore reliability of nucleotide detection. In one embodiment, the second and third oligonucleotides are linked so that, after nucleotide capture, they form a closed-loop single-stranded oligonucleotide component which is advantageously resistant to exonucleolysis.
As regards other prior art, Fan et al in Nature Reviews Genetics 7(8) 632-644 (2006) provide a general review of the development of methods and platforms that have enabled highly parallel genomic assays for genotyping, copy-number measurements, sequencing and detecting loss of heterozygosity, allele-specific expression and methylation. FIG. 2a of this review schematically shows the use of a circularizable probe with 3′ and 5′ ends that anneal upstream and downstream of a site of single nucleotide polymorphism (SNP) on an analyte thereby leaving a gap which is subsequently filled with a nucleotide which is the complement of the SNP to form a complete circular probe which may then be amplified after release. However, unlike our method, the nucleotide which is captured during the filling process is not obtained directly from the analyte itself.
WO03080861 discloses a process wherein a nucleic acid analyte is subjected to progressive pyrophosphorolysis in the presence of a nucleotide-specific reactive label which attaches directly to the nucleotide as it is released. Not only is this quite different from the method we employ but in practice the fluorescence signal measured when the labelled nucleotides are subsequently interrogated would likely be too weak to enable reliable identification above the associated background noise.
Finally, WO9418218 teaches a DNA sequencing method in which the analyte is subjected to progressive exonucleolysis to generate a stream of single nucleotide diphosphates or monophosphates which are then incorporated into a fluorescence-enhancing matrix before being detected. Not only is this a completely different approach to the one we describe but we again observe that any signal generated would likely be too weak to be reliably detected and identified.
We have now invented an improved method of generating an even stronger fluorescence signal which is suitable for use with the droplet-based sequencers we have previously described. Thus, according to the present invention there is provided a method of sequencing a nucleic acid characterised by the steps of (1) generating a stream of single nucleoside triphosphates by progressive enzymatic digestion of the nucleic acid; (2) producing at least one substantially double-stranded primary oligonucleotide used probe by reacting, in the presence of a polymerase and a ligase, at least one of the single nucleoside triphosphates with a corresponding primary probe comprising (a) a first single-stranded oligonucleotide including a first restriction endonuclease nicking-site, a single nucleotide capture site for capturing the single nucleoside triphosphate and oligonucleotide flanking regions juxtaposed either side of the capture site and (b) second and third single-stranded oligonucleotides capable of hybridising to the first oligonucleotide flanking regions; (3) nicking the first oligonucleotide strand of the used primary probe at the first nicking-site with a first nicking restriction endonuclease to create separate first oligonucleotide components; (4) separating the first oligonucleotide components generated in step (3) from the complementary strand of the used probe; (5) producing at least one substantially double-stranded secondary used probe by reacting, in the presence of a ligase, at least one of the separated first oligonucleotide components with a corresponding secondary probe comprising (c) a complementary fourth oligonucleotide including a second restriction endonuclease nicking-site and bearing fluorophores in a substantially undetectable state and optionally (d) a single-stranded fifth oligonucleotide at least in part complementary to the fourth oligonucleotide; (6) nicking the fourth oligonucleotide strand of the used secondary probe with a second nicking restriction endonuclease to create separate fourth oligonucleotide components at least some of which bear fluorophores in a detectable state and a single-stranded sixth oligonucleotide which is at least in part the sequence complement of the fourth oligonucleotide and (7) detecting the fluorophores released in step (6).
In one preferred embodiment, the complementary strand generated in step (4) is reacted with a further molecule of the first oligonucleotide and steps (3) and (4) are iterated in a first cycle as described below to increase significantly the number of fluorophores released for detection in step (7). In another embodiment the sixth oligonucleotide generated in step (6) is reacted with a further molecule of the fourth oligonucleotide and step (6) thereafter repeated to create a second cycle. Preferably the method of the invention involves iteration using both first and second cycles.
Step (1) of the method of the present invention comprises generating a stream of single nucleoside triphosphates from a nucleic acid analyte by progressive enzymatic digestion. In one embodiment this can be achieved by progressive exonucleolysis of the analyte followed by the action of a kinase on the single nucleoside monophosphates obtained (see for example Bao and Ryu, Biotechnology and Bioengineering DOI 10.1002/bit (May 2007)). In another embodiment step (1) comprises generating a stream of single nucleoside triphosphates directly from the analyte by progressive pyrophosphorolysis. The analyte employed in this step is suitably a double-stranded polynucleotide the length of which can in principle be unlimited; for example, including up to the many millions of nucleotide pairs found in a human gene or chromosome fragment. Typically, however, the polynucleotide will be at least 50, preferably at least 150 nucleotide pairs long; suitably it will be greater than 500, greater than 1000 and in many cases thousands of nucleotide pairs long. The analyte itself is preferably RNA or DNA of natural origin (e.g. derived from a plant, animal, bacterium or a virus) although the method can also be used to sequence synthetically-produced RNA or DNA or other nucleic acids made up wholly or in part of nucleotides whose nucleobases are not commonly encountered in nature; i.e. nucleobases other than adenine (A), guanine (G), cytosine (C), thymine (T) and uracil (U). Examples of such nucleobases include 4-acetylcytidine, 5-(carboxyhydroxylmethyl)uridine, 2-O-methylcytidine, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylamino-methyluridine, dihydrouridine, 2-O-methylpseudouridine, 2-O-methylguanosine, inosine, N6-isopentyladenosine, 1-methyladenosine, 1-methylpseudouridine, 1-methylguanosine, 1-methylinosine, 2,2-dimethylguanosine, 2-methyladenosine, 2-methylguanosine, 3-methylcytidine, 5-methylcytidine, N6-methyladenosine, 7-methylguanosine, 5-methylaminomethyluridine, 5-methoxyaminomethyl-2-thiouridine, 5-methoxyuridine, 5-methoxycarbonylmethyl-2-thiouridine, 5-methoxycarbonylmethyluridine, 2-methylthio-N6-isopentenyladenosine, uridine-5-oxyacetic acid-methylester, uridine-5-oxyacetic acid, wybutoxosine, wybutosine, pseudouridine, queuosine, 2-thiocytidine, 5-methyl-2-thiouridine, 2-thiouridine, 4-thiouridine, 5-methyluridine, 2-O-methyl-5-methyluridine and 2-O-methyluridine. In the case of DNA, the single nucleoside triphosphates generated are deoxyribonucleoside triphosphates whilst in the case of RNA they are ribonucleoside triphosphates.
In one embodiment of the method, step (1) further comprises a first sub-step of attaching the analyte to a substrate. Typically, this substrate comprises a microfluidic surface, a micro-bead or a permeable membrane; for example, one made of glass or a non-degradable polymer.
Preferably, the substrate further comprises a surface specifically adapted to receive the analyte. There are many ways in which the analyte can be attached to such surfaces any of which can in principle be used in this sub-step. For example, one method involves priming a glass surface with a functionalised silane such as an epoxysilane, an aminohydrocarbylsilane or a mercaptosilane. The reactive sites so generated can then be treated with a derivative of the analyte which has been modified to include a terminal amine, succinyl or thiol group.
In another embodiment of step (1), the analyte is pyrophosphorolysed to generate a stream of single nucleoside triphosphates the order of which corresponds to that of the sequence of the analyte. Such pyrophosphorolysis may be carried out at a temperature in the range 20 to 90° C.; for example, in the presence of a reaction medium comprising a suitable polymerase. Preferably it is carried out under conditions of continuous flow so that the single nucleoside triphosphates are continually removed from the reaction zone as they are liberated. Most preferably, the pyrophosphorolysis is carried out by causing an aqueous buffered medium containing the enzyme and the other typical additives to continuously flow over the surface to which the analyte is bound.
In yet another embodiment, the enzyme used is one which can cause progressive 3′-5′ pyrophosphorolytic degradation of the analyte to yield a stream of nucleoside triphosphates with high fidelity and at a reasonable reaction rate. Preferably this degradation rate is as fast as possible and in one embodiment is in the range 1 to 50 nucleoside triphosphates per second.
Further information about the pyrophosphorolysis reaction as applied to the digestion of polynucleotides can be found for example in J. Biol. Chem. 244 (1969) pp. 3019-3028. Suitably, the pyrophosphorolytic digestion is carried out in the presence of a medium which further comprises pyrophosphate anion and magnesium cations; preferably in millimolar concentrations.
In step (2) of the method of the present invention at least one single nucleoside triphosphate, preferably each single nucleoside triphosphate in the stream, is reacted in the presence of a polymerase and a ligase with a primary probe to generate a substantially double-stranded used primary probe. Preferably, before this step is carried out the product of step (1) is treated with an pyrophosphatase, to hydrolyse any residual pyrophosphate to phosphate anion.
Examples of polymerases which can be advantageously used include, but are not limited to, the prokaryotic pol 1 enzymes or enzyme derivatives obtained from bacteria such as Escherichia coli (e.g. Klenow fragment polymerase), Therm us aquaticus (e.g. Taq Pol), Bacillus stearothermophilus, Bacillus caldovelox and Bacillus caldotenax. Any suitable ligase can in principle be used in this step.
Each primary probe employed in step (2) is suitably comprised of (a) a first single-stranded oligonucleotide including a first restriction endonuclease nicking-site, a single nucleotide capture site for capturing the nucleoside triphosphate and oligonucleotide flanking regions juxtaposed either side of the capture site and (b) second and third single-stranded oligonucleotides capable of hybridising to the flanking regions. In one embodiment, the second and third oligonucleotides are discrete entities whilst in another they are linked to each other by means of a linker region. In this latter case and in one embodiment, the linker region links ends of the second and third oligonucleotides. The linker region can in principle be any divalent group but is conveniently another oligonucleotide region. In one embodiment this oligonucleotide linker region is unable to hybridise substantially to the first oligonucleotide.
The first, second and third oligonucleotides are chosen so that in step (2) the second and third oligonucleotides hybridise respectively to 3′ side and 5′ side flanking regions on the first oligonucleotide juxtaposed either side of a capture site. The capture site comprises the single nucleotide whose nucleobase is complementary to the one borne by the nucleoside triphosphate to be detected. This makes the three-component primary probe highly selective for that particular nucleoside triphosphate. For example, if the analyte is derived from DNA and the first, second and third oligonucleotides are comprised of deoxyribonucleotides, the capture site will be highly selective for deoxyadenosine triphosphate if the nucleotide it comprises bears a thymine nucleobase. Thus, in one useful embodiment of the invention step (2) may be carried out in the presence of a probe system comprised of a plurality of primary probe types; for example, one, two, three or four primary probe types each of which comprises a first oligonucleotide having different flanking regions and a different capture site characteristic of the various different nucleobases sought.
Typically, the first oligonucleotide is up to 150 nucleotides long, preferably between 10 and 100 nucleotides. In one embodiment the second oligonucleotide is shorter than the complementary 3′ side flanking region of the first oligonucleotide by at least one nucleotide. In another, there is a single nucleotide mismatch between the 3′ end of the first oligonucleotide and the nucleotide opposite it on the second oligonucleotide to prevent the nucleoside triphosphate being captured by the polymerase at this point. Similarly, in one embodiment the third oligonucleotide is longer than the complementary 5′ side flanking region of the first oligonucleotide by at least one nucleotide, while in another there is a single nucleotide mismatch between the 3′ end of the third oligonucleotide and the nucleotide opposite it in the first oligonucleotide to prevent the single nucleoside triphosphate being captured by the polymerase at this point.
In one preferred embodiment, the first nicking restriction endonuclease recognition site comprises an oligonucleotide region on the first oligonucleotide which itself includes the capture site. In another embodiment, where more than one primary probe type is employed, it is preferred that each first oligonucleotide nevertheless comprises a common first nicking restriction endonuclease recognition site so that only one first nicking restriction endonuclease need be employed. To achieve this, it is therefore preferred that the recognition site will be comprised of a sequence containing at least one of each of the typical nucleotides of RNA or DNA as the case may be.
In another preferred embodiment, the first nicking restriction endonuclease as defined herein is a conventional restriction endonuclease otherwise capable of cleaving both strands of the used primary probe and in the region of the nicking-site the strand created during the primary probe's use is rendered resistant to endonucleolysis; for example, by including one or more endonucleolytic blocking sites in the second and/or third oligonucleotides. In one embodiment, these blocking groups may be selected from phosphorothioate linkages and other backbone modifications commonly used in the art, C3 spacers, phosphate groups, or the like.
Step (2) is suitably carried out by contacting each single nucleoside triphosphate in the stream with the enzymes and one or more primary probe types described above at a temperature in the range 20 to 80° C.
The product of step (2) of the method of the invention is, as mentioned above, a substantially double-stranded used primary probe whose constituent strands are respectively the first oligonucleotide and a complementary oligonucleotide comprised of the second oligonucleotide, a nucleotide derived from the single nucleoside triphosphate and finally the third oligonucleotide. If the second and third oligonucleotides have previously been joined together by a linker region, then it will be readily apparent that this complimentary oligonucleotide will comprise a closed-loop strand.
In step (3) the used primary probe is treated with a first nicking restriction endonuclease at a temperature in the range 20 to 100° C. In this step the strand of the used primary probe derived from the first oligonucleotide is severed into two separate oligonucleotide components which can then in step (4) be de-hybridised from the probe's complementary strand thereby separating them from each other. Step (4) can be achieved by heating the nicked, used probe to a temperature in the range 30 to 100° C., but is most preferably achieved at the same temperature as that used in step (3).
In step (5) at least one of the separated oligonucleotide components is reacted in the presence of a ligase and a corresponding secondary probe to create at least one substantially double-stranded secondary used probe. In one embodiment, this secondary probe is comprised of a partially double-stranded fourth oligonucleotide bearing fluorophores in an undetectable state and the second restriction enzyme nicking-site and which is at least in part comprised of a single-stranded region which is the sequence complement of the relevant separated oligonucleotide component. In another, this fourth oligonucleotide is j-shaped as described in our application WO2014167323 to which the reader is directed for further information on its structural characteristics. In yet another embodiment the secondary probe comprises two components; (c) a single-stranded fourth oligonucleotide (including the second restriction endonuclease recognition site and the fluorophores in an undetectable state) which is at least in part comprised of a single-stranded region which is the sequence of one of the separated oligonucleotide components created in step (3) and (d) a single-stranded fifth oligonucleotide which is at least in part comprised of a single-stranded region which is the sequence complement of at least part of the fourth oligonucleotide.
In one preferred embodiment, where more than one secondary probe type is employed, it is preferred that each fourth oligonucleotide nevertheless comprises a common second nicking restriction endonuclease recognition site so that only one second nicking restriction endonuclease need be employed.
In one preferred embodiment the first and second restriction endonuclease recognition sites are the same, enabling a common first and second nicking restriction endonuclease to be employed in the method.
It is a feature of the fourth oligonucleotide that it is provided with a region which is labelled with its own unique type of fluorophores and that these fluorophore(s) are arranged so as to be substantially undetectable when the secondary probe is in an unused state. Preferably they are arranged to be essentially non-fluorescing at those wavelengths where they are designed to be detected. Thus, although a fluorophore may exhibit general, low-level background fluorescence across a wide part of the electromagnetic spectrum, there will typically be one or a small number of specific wavelengths or wavelength envelopes where the intensity of the fluorescence is at a maximum. It is at one or more of these maxima where the fluorophore is characteristically detected that essentially no fluorescence should occur. In the context of this patent, by the term ‘essentially non-fluorescing’ or equivalent wording is meant that the intensity of fluorescence of the total number of fluorophores attached to the fourth oligonucleotide at the relevant characteristic wavelength or wavelength envelope is less than 25%; preferably less than 10%; more preferably less than 1% and most preferably less than 0.1% of the corresponding intensity of fluorescence of an equivalent number of free fluorophores.
In principle, any method can be used to ensure that in the fourth oligonucleotide's unused state the fluorophore(s) are essentially non-fluorescing. In one embodiment this is achieved by disposing them in close proximity to quenchers. In another, it is achieved by arranging multiple fluorophores in close proximity to each other so that they tend to quench each other sufficiently well that quenchers are not required. In this context of this patent, what constitutes ‘close proximity’ between fluorophores or between fluorophores and quenchers will depend on the particular fluorophores and quenchers used and possibly the structural characteristics of the fourth oligonucleotide. Consequently, it is intended that this term should be construed with reference to the required outcome rather than any particular structural arrangement of these various elements. However, and for the purposes of providing exemplification only, it is pointed out that when adjacent fluorophores or adjacent fluorophores and quenchers are separated by a distance corresponding to the characteristic Forster distance (typically less than 5 nm) sufficient quenching will generally be achieved.
As regards the fluorophores themselves, they can in principle be chosen from any of those conventionally used in the art including but not limited to xanthene moieties e.g. fluorescein, rhodamine and their derivatives such as fluorescein isothiocyanate, rhodamine B and the like; coumarin moieties (e.g. hydroxy-, methyl- and aminocoumarin) and cyanine moieties such as Cy2, Cy3, Cy5 and Cy7. Specific examples include fluorophores derived from the following commonly used dyes: Alexa dyes, cyanine dyes, Atto Tec dyes, and rhodamine dyes. Examples also include: Atto 633 (ATTO-TEC GmbH), Texas Red™, Atto 740 (ATTO-TEC GmbH), Rose Bengal, Alexa Fluor™ 750 C5-maleimide (Invitrogen), Alexa Fluor™ 532 C2-maleimide (Invitrogen) and Rhodamine Red C2-maleimide and Rhodamine Green as well as phosphoramadite dyes such as Quasar 570. Alternatively, a quantum dot or a near infra-red dye such as those supplied by LI-COR Biosciences can be employed. The fluorophore is typically attached to the fourth oligonucleotide via a nucleobase using chemical methods known in the art.
Suitable quenchers include those which work by a Forster resonance energy transfer (FRET) mechanism. Examples of commercially available quenchers which can be used in association with the above mentioned-fluorophores include but are not limited to DDQ-1, Dabcyl, Eclipse, Iowa Black FQ and RQ, IR Dye-QC1, BHQ-0, BHQ-1, -2 and -3 and QSY-7 and -21.
In one embodiment at least one of the flanking regions of the first oligonucleotide also further comprises fluorophores as described above arranged so that the first oligonucleotide is substantially non-fluorescing in its unused state. In another embodiment at least one of the flanking regions further comprises quenchers.
The product of step (5) of the method of the invention is a substantially double-stranded used secondary probe comprised of the fourth oligonucleotide, the relevant oligonucleotide component and optionally the fifth oligonucleotide.
In one embodiment the fifth oligonucleotide or first oligonucleotide component is shorter than the complementary 3′ side flanking region of the fourth oligonucleotide by at least one nucleotide. In another, there is a single nucleotide mismatch between the 3′ end of the fourth oligonucleotide and the nucleotide opposite it on the fifth oligonucleotide or first oligonucleotide component to prevent the nucleoside triphosphate being captured by the polymerase at this point. Similarly, in one embodiment the fifth oligonucleotide or first oligonucleotide component is longer than the complementary 5′ side flanking region of the fourth oligonucleotide by at least one nucleotide, while in another there is a single nucleotide mismatch between the 3′ end of the fifth oligonucleotide or first oligonucleotide component and the nucleotide opposite it in the fourth oligonucleotide to prevent the single nucleoside triphosphate being captured by the polymerase at this point.
Thereafter in step (6) the used secondary probe is treated with the second nicking restriction endonuclease to create separate fourth oligonucleotide components. As explained above this second nicking restriction endonuclease can be identical to the first if the first and second nicking-sites are the same. Also it is envisaged that the second nicking restriction endonuclease can be a conventional restriction endonuclease as explained above if the relevant strand of the used secondary probe has been rendered resistant to endonucleolysis as explained above.
After this nicking has occurred the fourth oligonucleotide components are separated from the un-nicked complementary strand of the used secondary probe (hereinafter referred to as the sixth oligonucleotide) at which point the fluorophore(s) on these components become separated from each other or from the quencher(s) and become detectable. Thus as endonucleolysis of the used secondary probe proceeds, the observer sees the development of the fluorescence signal. The characteristics of this fluorescence then indirectly reflects the nature of the single nucleoside triphosphate originally captured by the primary probe.
It will be appreciated that steps (3) and (4) can be iterated in a first cycle by allowing a further first oligonucleotide to anneal to the complementary strand of the used primary probe subsequent to step (4), thus allowing the build-up of a high concentration of the relevant separated first oligonucleotide components and thereby allowing a corresponding high concentration of used secondary probes to be created. Also in another embodiment the sixth oligonucleotide generated in step (6) may be reacted with a further molecule of the fourth oligonucleotide and step (6) thereafter repeated to create a second cycle. Thus, when both the first and second cycles are iterated at the same time a double multiplier effect may be obtained. By this means, the fluorescence signal can be made to grow very quickly to a high intensity making its detection and identification easy.
Thereafter, and in step (7), the fluorophores liberated in step (6) are detected and the nature of the nucleobase attached to the single nucleoside triphosphate determined by inference. By carrying out the method of the invention systematically for all the single nucleoside triphosphates in an ordered stream generated in step (1), data characteristic of the sequence of original nucleic acid analyte can be generated and analysed. Methods of doing this are well-known in the art; for example, the reaction medium can be interrogated with light from a laser and any fluorescence generated detected using a photodetector or an equivalent device tuned to the characteristic fluorescence wavelength(s) or wavelength envelope(s) of the various fluorophores. This in turn causes the photodetector to generate a characteristic electrical signal which can be processed and analysed in a computer using known algorithms.
In one particularly preferred embodiment, the method of the present invention is carried out wholly or partially in a stream of microdroplets, at least some of which contain a single nucleoside triphosphate; suitably an ordered stream. Such a method may begin, for example, by inserting the nucleoside triphosphates generated in step (1) one-by-one into a corresponding stream of aqueous microdroplets maintained in an immiscible carrier solvent such as a hydrocarbon or silicone oil to help preserve the ordering. Alternatively, this can be achieved by directly creating the microdroplets downstream of the digestion (pyrophosphorolysis) zone; for example, by causing the reaction medium to emerge from a microdroplet head of suitable dimensions into a flowing stream of the solvent. Alternatively, small aliquots of the reaction medium from step (1) can be regularly and sequentially injected into a stream of pre-existing aqueous microdroplets suspended in the solvent. If this latter approach is adopted, each microdroplet may already contain the various components of the primary and secondary probes together with the enzymes and any other reagents (e.g. buffer) required to effect steps (2) to (6). In yet another approach, the microdroplets created in the former embodiment can be caused to coalesce subsequently with a stream of such pre-existing microdroplets to achieve a similar outcome. In these microdroplet methods, step (7) then preferably involves delivering the microdroplets to a storage area and interrogating each microdroplet to identify the fluorophores liberated. Thereafter the results obtained from each microdroplet are assembled into a stream of data characteristic of the sequence of the original nucleic acid analyte.
To avoid the risk that a given microdroplet contains more than one nucleoside triphosphate it is preferred to release each nucleoside triphosphate in step (1) at a rate such that each filled microdroplet is separated on average by from 1 to 20 preferably 2 to 10 empty ones. Thereafter the stream of filled and unfilled microdroplets in the solvent is caused to flow along a flow path, suitably a microfluidic flow path, at a rate and in a manner such that they are maintained in a discrete state and do not have the opportunity to coalesce with each other. Suitably the microdroplets employed have a finite diameter less than 100 microns, preferably less than 50 microns, more preferably less than 20 microns and even more preferably less than 15 microns. Most preferably of all their diameters are in the range 2 to 20 microns. In one embodiment, the microdroplet flow rate through the whole system is in the range 50 to 3000 microdroplets per second preferably 100 to 2000.
In a second aspect of the present invention there is provided a multi-component biological probe system characterised by including (1) a primary probe comprising (a) a first single-stranded oligonucleotide including a first restriction endonuclease nicking-site, a single nucleotide capture site for capturing a single nucleoside triphosphate and oligonucleotide flanking regions juxtaposed either side of the capture site and (b) second and third single-stranded oligonucleotides capable of hybridising to the flanking regions and (2) a secondary probe comprising (c) an at least partially single-stranded fourth oligonucleotide bearing fluorophores in a substantially undetectable state having a single-stranded region complementary to at least part of the first oligonucleotide and including a second restriction endonuclease nicking-site and (d) optionally a single-stranded fifth oligonucleotide at least in part complementary to the fourth oligonucleotide.
In one embodiment the second and third oligonucleotides are connected by a linker region; for example, an oligonucleotide region. In another the first restriction endonuclease nicking-site is an oligonucleotide region including the capture site. In another embodiment the first and second restriction endonuclease nicking sites are the same. In yet another embodiment the fourth oligonucleotide is either (1) partially double-stranded, i.e. j-shaped, or (2) single-stranded and used in association with the fifth oligonucleotide.
Suitably, the primary probe comprises from one to four different first oligonucleotide types differing in the sequences of their flanking regions and in the nucleotide characteristic of the capture region. In another embodiment the secondary probe comprises from one to four different fourth oligonucleotide types differing in the fluorophores they bear and in their sequences.
Preferably the probe system further comprises at least one of a ligase, a polymerase and first and second nicking restriction endonucleases capable of nicking the first and second nicking-sites once the primary and secondary probes have been used.
Details of suitable nicking restriction endonucleases which can be used with the method, the primary and secondary probes and probe systems of the present invention can be found at http://rebase.neb.com in the database associated therewith.
It is believed that the detection method used in the sequencing method described above is also generally applicable to the analysis, characterisation or quantification of single nucleoside triphosphates in a biological sample or an analyte derived therefrom. Thus, in a third aspect of the present invention, there is provided a method of analysing a single nucleoside triphosphate characterised by the steps of (1) producing at least one substantially double-stranded primary oligonucleotide used probe by reacting, in the presence of a polymerase and a ligase, the single nucleoside triphosphate with a corresponding primary probe comprising (a) a first single-stranded oligonucleotide including a first restriction endonuclease nicking-site, a single nucleotide capture site for capturing the single nucleoside triphosphate and oligonucleotide flanking regions juxtaposed either side of the capture site and (b) second and third single-stranded oligonucleotides capable of hybridising to the first oligonucleotide flanking regions; (2) nicking the first oligonucleotide strand of the used primary probe at the first nicking-site with a first nicking restriction endonuclease to create separate first oligonucleotide components; (3) separating the first oligonucleotide components generated in step (2) from the complementary strand of the used probe; (4) producing at least one substantially double-stranded secondary used probe by reacting, in the presence of a ligase, at least one of the separated first oligonucleotide components with a corresponding secondary probe comprising (c) a complementary fourth oligonucleotide including a second restriction endonuclease nicking-site and bearing fluorophores in a substantially undetectable state and optionally (d) a single-stranded fifth oligonucleotide at least in part complementary to the fourth oligonucleotide; (5) nicking the fourth oligonucleotide strand of the used secondary probe with a second nicking restriction endonuclease to create separate fourth oligonucleotide components at least some of which bear fluorophores in a detectable state and a single-stranded sixth oligonucleotide which is at least in part the sequence complement of the fourth oligonucleotide and (6) detecting the fluorophores released in step (5).
In such a method the nature of the various steps and the biological probes employed will suitably be as described above. In one embodiment the single nucleoside triphosphate will be derived from a precursor double-stranded DNA analyte by pyrophosphorolysis. In another it will be generated from a precursor single nucleoside monophosphate or single nucleoside diphosphate using for example a kinase (see for example Biotechnology and Bioengineering by Bao and Ryu (DOI 10.1002/bit.21498)).
The invention is now illustrated with reference to the following Examples.
A single-stranded first oligonucleotide 1 was prepared, having the following nucleotide sequence:
wherein A, C, G, and T represent nucleotides bearing the relevant characteristic nucleotide base of DNA. It further comprises a capture region (A nucleotide) at the 13th base from its 5′ end, selective for capturing deoxythymidine triphosphate nucleotides (dTTPs) in a mixture of deoxynucleoside triphosphates (dNTPs), and the recognition sequence for the nicking restriction endonuclease Nb.BsrDI, ‘NNCATTGC’.
Another single-stranded oligonucleotide 2, comprising an oligonucleotide region having a sequence complementary to the 3′ flanking region of the first oligonucleotide, and a single-stranded oligonucleotide 3, comprising an oligonucleotide region having a sequence complementary to the 5′ flanking region of the first oligonucleotide with a one base mismatch at its 3′ end and a 5′ phosphate group, were also prepared. They had the following nucleotide sequences:
wherein P represents the 5′ phosphate group.
A single-stranded fourth oligonucleotide was also prepared, having the following nucleotide sequence:
wherein X represents a deoxythymidine nucleotide (T) labelled with Atto 594 dye using conventional amine attachment chemistry and Q represents a deoxythymidine nucleotide labelled with a BHQ-2 quencher. It further comprises a 3′ region complementary to the 5′ flanking region of the first oligonucleotide, and the recognition sequence for the nicking restriction endonuclease Nb.BsrDI, ‘NNCATTGC’.
A single-stranded fifth oligonucleotide was also prepared, having the following nucleotide sequence, which is substantially complementary to the 5′ region of the fourth oligonucleotide:
A reaction mixture comprising the probe system was then prepared. It had a composition corresponding to that derived from the following formulation:
20 uL 5× buffer pH 7.9
10 uL oligonucleotide 1, 1000 nM
3 uL oligonucleotide 2, 100 nM
5 uL oligonucleotide 3, 100 nM
10 uL oligonucleotide 4, 1000 nM
5 uL oligonucleotide 5, 100 nM
10 uL spermine solution, 10 mM
10 U Nb.BsrDI nicking endonuclease (ex. New England Biolabs Inc.)
80 U Taq DNA Ligase
5.8 U Bst Large Fragment polymerase
6.7 U Thermostable Inorganic Pyrophosphatase
2.5 uL mixture of dNTPs, 1 nM
Water to 100uL
wherein the 5× buffer comprised the following mixture:
25 uL Trizma Acetate, 1M, pH 7.9
50 uL aqueous Magnesium Acetate, 1M
25 uL aqueous Potassium Acetate, 1M
50 uL Triton X-100 surfactant (10%)
500 μg BSA
Water to 1 mL
Capture of the dTTPs and ligation of oligonucleotide 2 to oligonucleotide 3 to form a used primary probe was then carried out by incubating the mixture at 37° C. for 30 minutes after which the temperature was increased to 56° C. for a further 15 minutes to allow iterated nicking of the first oligonucleotide. The temperature was then reduced to 37° C. for a further 30 minutes to allow ligation of the resulting first oligonucleotide components to the fifth oligonucleotide against the fourth oligonucleotide to form completed secondary probes. The temperature was then increased to 56° C. for 90 minutes to allow iterated nicking of the fourth oligonucleotide. The reaction mixture was optically excited and the resulting characteristic fluorescence of the Atto 594 dye detected using a CLARIOstar microplate reader (ex BMG Labtech) as the cycles of endonucleolysis occurred.
The growth in intensity of fluorescence over time in the presence and absence of the dNTP component of the reaction was monitored and the results shown graphically in
An aqueous medium 1 comprising a stream of single nucleotide triphosphates obtained by the progressive pyrophosphorolysis of a 100 nucleotide base polynucleotide analyte derived from human DNA is caused to flow through a ten-micron diameter microfluidic tube fabricated from PDMS polymer. The pyrophosphorolysis reaction itself is carried out by passing a stream of an aqueous, buffered (pH 7.5) reaction medium at 72° C., comprising Taq Pol and a solution having a 2 millimoles per litre concentration of each of sodium pyrophosphate and magnesium chloride, over a glass micro bead onto which the analyte has been previously attached by means of a succinyl bridge. The order of the single nucleotides in 1, which is downstream of the micro bead, corresponds to the sequence of the analyte. 1 emerges from a droplet head 2 into a first chamber 3 where it is contacted with one or more streams of immiscible light silicone oil 4. The velocities of these streams are chosen to avoid turbulent mixing and to create aqueous spherical droplets 5 suspended in the oil each having a diameter of approximately eight microns. Typically, rates are adjusted so that between adjacent filled droplets there are on average 10 empty ones. A stream of 5 is then carried forward along a second microfluidic tube of the same diameter to a second chamber 6 into which a second stream of five micron aqueous spherical droplets 7 is also fed by means of a second droplet head 8. Droplets 5 and 7 are caused to coalesce in a sequential fashion to form enlarged aqueous droplets 9 approximately nine microns in diameter. Each of 7 contains inorganic pyrophosphatase to destroy any residual pyrophosphate anion present in each of 5.
A stream of 9 is then carried forward at the same rate via microfluidic tubing into a third chamber 10 where these droplets are contacted with a third stream of five micron aqueous spherical droplets 11 also fed thereto through a corresponding droplet head 12. The time taken for each of 9 to move between chambers 6 and 10 is c.2minutes.
Droplets 9 and 11 are then caused to coalesce in 10 to produce droplets 13 (approximately ten microns in diameter). Each of 11 contains a mesophilic ligase, a thermophilic polymerase, the nicking restriction endonuclease Nb.BsrDI (ex. New England Biolabs Inc.), a primary probe comprising four sets of single-stranded oligonucleotides similar to those described in Example 1 and a secondary probe comprising four different fourth single-stranded oligonucleotide labelled with a different fluorophore and four complementary fifth oligonucleotides. Alternatively, these components may be added in a series of coalescence steps (not shown) in which different droplet types 11, each containing one or more of these components, are coalesced in turn with 9 or a droplet arising from a previous coalescence to eventually yield 13.
The stream of the coalesced microdroplets 13 so formed is then subjected to incubation at 37° C. for 30 minutes followed by 56° C. for 15 minutes followed by 37° C. for 30 minutes followed by 56° C. for 90 minutes. At the end of this time 13 is transferred to the detection system, 14.
The detection system (not shown) typically comprises a detection window in which each droplet is interrogated with incident light from a laser. Action of this light then causes the released fluorophores in each droplet to fluoresce in a way characteristic of the single nucleotide base which was originally incorporated into the primary probe (or essentially not at all if the droplet was originally empty). The presence or absence of this fluorescence is then detected at the four characteristic wavelengths of the four fluorophores associated with the four oligonucleotide sets mentioned above. Thus as the droplets are interrogated in turn the sequence of nucleotide bases in the original polynucleotide analyte can in effect be read off.
| Number | Date | Country | Kind |
|---|---|---|---|
| 16189791.3 | Sep 2016 | EP | regional |
| 1618920.1 | Nov 2016 | GB | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2017/073759 | 9/20/2017 | WO | 00 |
