WO2014033285A1 - Méthode de séquençage pour polynucléotide monobrin utilisant une sonde avec site de reconnaissance pour nucléase - Google Patents

Méthode de séquençage pour polynucléotide monobrin utilisant une sonde avec site de reconnaissance pour nucléase Download PDF

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Publication number: WO2014033285A1
Authority: WO; WIPO (PCT)
Prior art keywords: polynucleotide; probe; complementary; test; recognition
Prior art date: 2012-08-30
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.): Ceased

Application number

PCT/EP2013/068038

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English (en)

Inventor

Eirik SELSAAS

Jarle Kotsbak

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GENESEQUE AS

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GENESEQUE AS

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2012-08-30

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2013-08-30

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2014-03-06

2013-08-30 Application filed by GENESEQUE AS filed Critical GENESEQUE AS

2014-03-06 Publication of WO2014033285A1 publication Critical patent/WO2014033285A1/fr

2015-02-28 Anticipated expiration legal-status Critical

Status Ceased legal-status Critical Current

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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6869—Methods for sequencing

Definitions

the present invention concerns an improved sequencing method, to prevent stalling of a sequencing reaction and/or to achieve or improve the synchronisation of a multimolecular sequencing reaction.
the invention relates to the use of a recognition probe which binds to a single stranded polynucleotide sequence and which has a recognition site for a nuclease whose cleavage site is separate from the recognition site. Sequencing of part of the polynucleotide is carried out using standard sequencing techniques by the incorporation of complementary test bases or test probes to generate a complementary nucleotide strand of the polynucleotide sequence.
the nuclease that recognises the site in the probe is used to cleave the polynucleotide and its complementary nucleotide strand. This generates polynucleotides of the same length and/or with the same terminal sequence in a multimolecule sequencing reaction which can be used in a further sequencing cycle.
the method thus synchronises a multimolecule reaction which may have become desynchronised and will enable a stalled reaction in either a multimolecule or single molecule sequencing reaction to be continued.
Polynucleotide sequencing has been carried out by various methods for many years and has provided a mine of information concerning the genomes of different species. Even though the human genome has been sequenced, there is a great interest in resequencing in order to identify genetic predispositions or genetic or gene related diseases. Hence, the mapping of mutations and tissue specific mRNA production and expression analysis is of great interest. Additionally, the sequencing of genomes from other species is of interest i.e. de novo sequencing.
DNA sequencing has been traditionally carried out by the Sanger dideoxy method (Sanger, Nicklen and Coulson, Proc. Natl. Acad. Sci. USA, 1977, 74, 5463- 7) which has been used since the 1980s. It is a multimolecular method based on electrophoretic filtering of cloned DNA that is firstly treated enzymatically. The enzymatic process produces single stranded DNA by interrupted polymerisation using a mixture of fluorophore labelled dideoxy NTPs which terminate chain extension and dNTPs which do not.
the chain termination method of Sanger therefore requires the generation of one or more sets of labelled DNA fragments, which each terminate with a particular nucleotide base.
the fragments must be separated by size to determine the sequence and thus the electrophoretic gels used must be able to distinguish large fragments which differ in size by a single nucleotide. This limits the size of the DNA chain that can be sequenced at one time.
Realtime methods include sequencing by synthesis which can be used for multimolecule or single molecule sequencing and sequencing by ligation (for multimolecule sequencing). Sequencing by synthesis involves the use of fluorophore labelled terminating nucleotide bases which are added to an
a single terminating nucleotide base is thus incorporated by polymerisation into the target DNA sequence in each cycle and the base is then determined by virtue of its fluorophore label.
the terminating base can be chemically neutralised once the readout has been obtained to allow
the lipid chain between the base and the label can be cleaved chemically or photochemically so that previously incorporated labels can be removed to allow the reading of subsequently incorporated labels.
Sequencing by ligation involves ligating fluorophore labelled probes to an unknown sequence where the sequence can be determined by the sequence of the probe which is able to ligate thereto.
One commonly used method for sequencing by ligation involves four fluorescently labelled di-base probes competing for ligation to a sequencing primer (Applied Biosystems). Every first and second base in each ligation reaction is evaluated by the probes. After each ligation reaction, the fluorescent label is read and subsequently cleaved from the probe and a further ligation reaction is carried out with the probe set. Once a complete series of ligation reactions has been carried out, the extended strand is melted away and a new ligation reactions series is performed using a primer complementary to the n-1 position. Five rounds of primer reset may be carried out using this method to determine the sequence.
limitations can occur on the read length of a target sequence using prior art methods of sequencing (e.g. sequencing by synthesis and sequencing by ligation), in view of the fading of the signal, caused by more and more strands being stalled or becoming asynchronous. For multimolecular methods, this results in fewer strands sending out the signal from the correct base to be sequenced.
Desynchronisation is caused by dephasing of which there are two forms.
the first form is lagging and may result from the inefficiency of DNA polymerase when incorporating labelled nucleotides in a sequencing by synthesis method (see Figure 1 ). If a nucleotide is not added to a strand in a sequencing cycle, this strand will become one nucleotide behind the other strands and this will yield signals from the base that is one cycle behind the other strands.
the second form of dephasing in sequencing by synthesis is read ahead which is where a nucleotide without a termination is added (in methods where terminating nucleotides are used).
the present invention represents a modification to sequencing methods of the art e.g. sequencing by synthesis, sequencing by ligation, Pyrosequencing and Ion semiconductor sequencing, which use nucleotides or probes in the sequencing reactions (and particularly methods which use modified nucleotides or probes), and improves such sequencing methods by preventing desynchronisation and/or stalling.
the present invention provides a method for determining a nucleotide sequence of a polynucleotide comprising the steps of: 1 ) providing a single stranded polynucleotide;
identifying one or more nucleotides in the polynucleotide by determining which at least one complementary test base or which at least one test probe bound to the polynucleotide sequence in step 3);
nuclease which recognises the recognition site in the recognition probe bound to said polynucleotide, wherein the nuclease cleaves the polynucleotide at a site which is double stranded from the binding of said complementary test bases or said test probes to the polynucleotide sequence during step 3) or at a site on the polynucleotide which is single stranded and which is downstream of said bound complementary test bases or said bound test probes;
each cycle of steps 2) to 6) is carried out one or more times until the polynucleotide sequence has been determined and wherein when a
step 3) may precede step 2).
the nuclease referred to in step 6) is capable of cleaving only double stranded nucleic acid molecules or is capable of cleaving both double stranded and single stranded nucleic acid molecules.
the present invention relates to an improved method of polynucleotide sequencing, wherein a recognition probe is used to bind to a single stranded polynucleotide sequence which recognition probe has a recognition site for a nuclease e.g. a type II or III restriction enzyme.
a complementary nucleotide strand is then extended from the recognition probe or from a primer bound to the recognition probe and/or polynucleotide which allows one or more nucleotides in the polynucleotide to be determined.
a nuclease e.g. a type II or III restriction enzyme
the nuclease will cleave the polynucleotide and the generated complementary nucleotide strand i.e. the nuclease will act on a double stranded molecule and will cleave this molecule.
the formation and extension of the complementary nucleotide strand generates the cleavage site for the enzyme by generating a double stranded polynucleotide (comprising the original single stranded polynucleotide and its complementary nucleotide strand) upon which the enzyme can act.
an enzyme which can additionally (or alternatively) cleave single stranded polynucleotide
an enzyme may cleave the polynucleotide at a particular position regardless of whether any complementary nucleotide strand is present at that position.
Such an enzyme may therefore cleave the polynucleotide at a site which is downstream of the bound complementary nucleotide strand.
the enzyme may cleave both sequenced and hence synchronised double stranded polynucleotides and also lagging strands which may be single stranded at that position (which obviates the requirement to fill in lagging strands for nuclease cleavage).
the enzyme may cleave non-lagging and lagging strand at the same time, without any requirement to make the lagging strands double stranded at the site of cleavage.
the cleavage of the polynucleotide results in all strands being cut at a specific distance from the recognition site which produces polynucleotide strands with the same terminal 3' sequence and/or which may be of the same length, which can then be used for another sequencing cycle (the next sequencing reaction).
This method therefore ensures that any strands which are desychronised or stalled in the first sequencing cycle are all cleaved at the same position as strands which are synchronised in that cycle (assuming extension beyond the cleavage site is achieved when using enzymes which solely cleave double stranded DNA).
the cleavage step should therefore preferably result in the production of a collection of polynucleotide strands cleaved at the same position and/or of the same length.
a further sequencing reaction or cycle can then be carried out until another cleavage reaction is initiated (which may be by the generation of a double stranded cleavage site), when again the polynucleotides may be cut.
This method of sequentially sequencing and cleaving polynucleotides allows the synchronisation and unstalling of the sequencing reaction. For single molecule sequencing, the method may allow the sequencing of a stalled strand to be reinitiated.
the invention provides a method for determining a nucleotide sequence of a polynucleotide comprising the steps of:
identifying one or more nucleotides in the polynucleotide by determining which at least one complementary test base or which at least one test probe bound to the polynucleotide sequence in step 3);
each cycle of steps 2) to 6) is carried out one or more times until the polynucleotide sequence has been determined and wherein when a
step 3) may precede step 2).
multimolecule sequencing is carried out i.e. sequencing of multiple copies of a polynucleotide (i.e. more than 1 copy e.g. more than 2, 10, 100, 1000 or 10000 copies).
a step of melting is carried out after cleavage step 6) to remove any of the complementary nucleotide strand which is still hybridised to the polynucleotide after cleavage (of a double stranded molecule) to provide a single stranded polynucleotide.
cleavage of the polynucleotide and its bound complementary nucleotide strand generally results in a small portion of the complementary strand remaining on the polynucleotide, in view of the complementary nucleotide strand being extended past the formation of the cleavage site and in view of the staggered cleavage sites of nuclease enzymes on the 5' to 3' and 3' to 5' strands i.e. nuclease enzymes generally cleave leaving sticky ends on polynucleotides.
nuclease enzymes generally cleave leaving sticky ends on polynucleotides.
complementary nucleotide strand refers to the nucleotide strand formed by the incorporation (e.g. by polymerisation or ligation) of a complementary test base(s) or test probe(s) on the single stranded polynucleotide.
complementary nucleotide strand is complementary to at least part of the polynucleotide whose sequence is to be determined.
sequence of the polynucleotide is determined by sequential polymerase/ligation and cleavage steps, the complementary nucleotide strand is usually only formed for a part or portion of the polynucleotide during any particular sequencing cycle.
the complementary nucleotide strand is therefore typically shorter than the polynucleotide e.g. may be less than 5, 10, 15, 25, 30, 35, 40, 45, 50, or 100 nucleotides in length or alternatively viewed may be less than 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50% of the length of the polynucleotide and is only complementary to a part of the
the portion of complementary nucleotide strand remaining bound to the polynucleotide after cleavage is only a part of the complementary nucleotide strand generated in the sequencing cycle by the incorporation of a complementary test base(s) or test probe(s).
the portion of complementary nucleotide strand remaining is less than 5, 10, 15, 20, 30, 40 or 50% of the length of the generated complementary nucleotide strand.
the melting step may be carried out either before or after the step of contacting the polynucleotide with the recognition probe in the next sequencing cycle e.g. in step 1 ) or 2). In the latter case, the melting step may be carried out during or after step 2).
the melting step i.e. the removal of the portion of complementary nucleotide strand remaining bound to the polynucleotide after cleavage, is preferably carried out by increasing the temperature and/or by reducing the salt concentration. The increased temperature required to remove the portion of the complementary nucleotide strand depends on the length of the portion of the complementary nucleotide strand remaining.
the melting step performed therefore results in the production of single stranded polynucleotides which are preferably the same length and which can then be subjected to a further sequencing cycle by removing any remaining portions of complementary nucleotide strands.
the cleavage step 6) which may digest the polynucleotide and its complementary nucleotide strand produced by the sequencing reaction, usually results in a portion of the complementary nucleotide strand being left bound to the polynucleotide (see Figure 2).
the portions of the complementary strands remaining bound to different polynucleotides may be of different lengths.
the sequencing reaction of one particular polynucleotide strand has become desynchronised by lagging and has become 1 or more bases behind the
the complementary nucleotide strand formed in this reaction will be 1 or more bases shorter than for other strands.
the portion of complementary nucleotide strand remaining will be shorter than the other strands.
the portion of the complementary nucleotide strand remaining after cleavage of a double stranded molecule may advantageously increase ligation efficiency of the recognition probe.
the melting step for removal of the portion of the complementary nucleotide strand can thus be carried out after or during the step of contacting the recognition probe with the polynucleotide.
step 2) of the method of the invention may include an additional step of melting to remove any complementary nucleotide strand remaining after cleavage.
the method may encompass repeating steps 2) to 4) and 6) (and optionally step 5)) one or more times until the polynucleotide sequence has been determined, wherein a step of melting is carried out during or after step 2).
the melting step may be carried out during or after the addition of the recognition probe, regardless of when the complementary test bases/test probes are added.
the melting step is carried out during or after the step of contacting recognition probe with the polynucleotide to improve ligation efficiency, then it is possible that the recognition probe strand bound or hybridised to the polynucleotide may also be removed. As discussed in detail below, this strand of the recognition probe may comprise an overhang or protrusion which is capable of hybridising with the single stranded polynucleotide. If one strand of the recognition probe (usually the strand comprising any single stranded overhang or alternatively viewed the strand which may be extended to form the complementary nucleotide strand) is removed in the melting step, then a further step of binding a primer complementary to the remaining probe strand may be required.
the recognition probe may be produced having cross-linked strands to prevent removal by melting i.e. during the melting step to remove any portion of the complementary nucleotide strand.
the recognition probe may be "self-priming", where the recognition probes may be single stranded but designed to form hairpin structures allowing the probe to act as double stranded DNA. Probes that form hairpin structures in this way allow the primer strand of the recognition probe to remain associated with the probe under any conditions and are referred to herein as "self-priming".
recognition probe to either have cross-linked strands or to be self-priming would prevent the melting of the recognition probe strand which is capable of being extended to form the complementary nucleotide strand and would abrogate the requirement to carry out the additional step of binding a primer complementary to the remaining recognition probe strand.
a melting step may be carried out after step 4 (or 5)) but before step 6) of the method of the invention i.e. after the identification of at least one nucleotide in the polynucleotide but prior to the cleavage step.
This melting step will remove the complementary nucleotide strand formed in order to identify the at least one nucleotide in the polynucleotide, which complementary nucleotide strand may comprise one or more modified nucleotides or probes.
a further step would then be carried out of generating a complementary nucleotide strand using non-modified nucleotides.
nucleotides may include natural or non-natural nucleotides as discussed below but do not have any modification e.g. non-modified nucleotides do not comprise a signalling means or have any other entity attached thereto.
the generation of this strand would not allow sequencing of the polynucleotide (one or more nucleotides would have been previously identified by the incorporation of one or more modified complementary test bases/test probes), but may be carried out when using nucleases in the invention which cleave only double stranded nucleic acids and which are not able to cleave at sites containing modified bases. When using such an enzyme it is thus necessary to remove any modified bases and replace with non-modified bases before carrying out the cleavage step.
nuclease used to cleave the polynucleotide and its complementary nucleotide strand in the present invention is capable of cleaving at sites or in polynucleotides having or comprising modified bases or nucleotides.
a step of unbound recognition probe removal may be carried out after contacting the recognition probe with the polynucleotide.
Recognition probes may comprise labels or be conjugated to other entities to allow the removal of unbound probe as discussed further below.
the present invention will achieve synchronisation in a sequencing reaction by sequentially generating a complementary nucleotide strand and cleaving the polynucleotide to produce polynucleotide strands with identical end sequences and/or length for further sequencing cycles.
further additional steps may be carried out in the method to ensure synchronisation.
a step of modification of the 3' ends of the polynucleotide strands after contacting said polynucleotide strand with the recognition probe.
This step may prevent polynucleotides which have not bound to a recognition probe from being able to take part in any subsequent ligation reactions. Thus, such polynucleotides, would not be capable of binding to a recognition probe in any subsequent sequencing cycle.
This step hence prevents such polynucleotides from being desynchronised and from being able to produce a desynchronised sequencing signal.
determining a nucleotide sequence refers to the determination of a partial as well as a full sequence. (This phraseology is used interchangeably with "identifying" a base/nucleotide or bases/nucleotides in a sequence.) Any sequence length is encompassed by the determination of a nucleotide sequence, hence, at least one nucleotide base may be determined by the method, although preferably more than one nucleotide may be determined e.g. at least 2, 3, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 4000, 6000 or 10000 or more nucleotides may be
determining a nucleotide sequence refers to the identification of at least one nucleotide in the polynucleotide.
a sequencing cycle carried out in the method of the invention it is possible that only one of the nucleotides is identified in the polynucleotide, even if the complementary nucleotide strand generated in that sequencing cycle is more than one nucleotide in length.
At least 10, 20, 30, 40, 50, 60, 70, 80 or 90% of the nucleotides in the portion of the polynucleotide to which the complementary nucleotide strand has been generated are identified in a sequencing cycle.
a sequencing cycle refers to each performance of steps 2) to 4) and 6) (and optionally step 5)) of the invention.
steps 2) to 4) and 6) are one sequencing cycle and one or multiple cycles may be carried out to determine the polynucleotide sequence.
step 6) is necessary only to prepare the polynucleotide for the next cycle and this may not be performed in the final cycle.
a sequencing reaction refers to the incorporation of one of more
Determination of the nucleotide sequence includes the identification of the specific base at a particular position (i.e. A, T/U, G or C), i.e. absolute identification, or provides partial identification of that base, e.g. the method may identify a set of bases, of less than 4, (i.e. 3 or 2) which consists of the options for that base, e.g. A or T, but not G or C, or A, T or G but not C.
the partial identification provides information on the identity of the base which when coupled with information obtained, e.g. in other cycles, allows absolute identification of the base.
Such partial identification in a cycle is especially useful when more than one base is to be identified (i.e. read) in each cycle, i.e.
test probe set which is not fully degenerate.
a combination of the information obtained from overlapping readings may be used to obtain the sequence unambiguously or close enough to unambiguously to be useful, especially when the individual that is the source of the sequencing material belongs to a species for which the genome mapping is known, and primarily single nucleotide polymorphisms (SNP) data is the aim of the sequencing.
SNP single nucleotide polymorphisms
the fact that each base will be involved in at least two reading cycles may, with the right combination of test probe sequences, be used to enhance the information level to increase the data quality.
determining a nucleotide sequence includes resequencing known nucleotide sequences, as well as sequence comparisons and investigating polymorphisms and mutations in known sequences. Additionally, "determining a nucleotide sequence” may encompass determining the positions of one, two or three of the four types of nucleotides in a sequence, for example, it may be desirable to only determine the position of cytosines within a sequence, as well as identifying the positions in the sequence of any or all of the four nucleotide bases.
nucleotide base in the polynucleotide sequence is determined by the invention.
the sequencing reaction in each cycle will usually continue until the polynucleotide is double stranded at the site of cleavage by the nuclease.
the sequencing reaction and the generation of the complementary nucleotide strand results in the formation of the cleavage site. This is particularly the case when the nuclease can only digest double stranded nucleic acid molecules.
nucleases are available that cleave or digest polynucleotides at least 5, 10, 15 or 20 nucleotide bases from their recognition sites.
the sequencing reaction of each cycle allows the production of a complementary nucleotide strand that is at least 5, 10, 15 or 20 nucleotides in length and is of a length which allows cleavage by a nuclease.
the strand may be shorter in length depending on the distance of the recognition site in the probe from the start of the polymerisation reaction and hence the beginning of the production of the complementary strand.
the sequencing reaction may necessarily produce a complementary nucleotide strand of at least 5, 10, 15 or 20 nucleotides, as indicated above, it is not necessary that all of the nucleotides in the complementary positions in the polynucleotide are identified. Therefore, it is possible that only one of the nucleotides is identified, even though a much longer complementary nucleotide strand is produced.
the complementary nucleotide strand generated may be longer than at least 5, 10, 15 or 20 nucleotides. It is desired to allow the incorporation of complementary test bases/test probes into the complementary nucleotide strand beyond the cleavage site, to ensure that all polynucleotide strands are of sufficient length to allow digestion/cleavage by the nuclease particularly when using a nuclease which only digests a double stranded nucleic acid. Thus, when using a nuclease which only digests double stranded nucleic acid molecules it is necessary that, for example, desynchronised strands reach the necessary length to comprise a cleavage site. However, the
complementary strand should preferably only be made long enough to ensure cleavage of all or most strands, as longer complementary strands are more difficult to remove from the polynucleotide by melting.
cleavage of the polynucleotide should be carried out after each sequencing cycle and the generation of a cleavage site particularly when using a nuclease which only digests double stranded nucleic acids.
nuclease which may cleave single stranded as well as double stranded nucleic acid molecules at a particular distance from its recognition site, then extension of the complementary nucleotide strands beyond the cleavage site may not be necessary.
strands which are synchronised will be double stranded at the cleavage site and the bases of interest may have been sequenced. Any lagging strands which may be 1 , 2, 3 or more bases/probes behind the synchronised sequencing reaction may be single stranded at the cleavage site (depending on how far past the cleavage site the reaction has run).
nuclease which is being used can cleave both the synchronised (or non-lagging strands) and the lagging strands, which may be single stranded at the cleavage site, then there is no need to extend the complementary nucleotide strands beyond the cleavage site or to ensure that the lagging strands are double stranded at the cleavage site. All strands whether lagging and potentially single stranded at the cleavage site or non-lagging and double stranded at the cleavage site, will be capable of being cleaved with such a nuclease and thus of being synchronised.
polynucleotide whose sequence is determined in the method of the invention may be any polynucleotide but is preferably a DNA or RNA sequence.
RNA sequences are subjected to reverse transcription to produce copy DNA before being subjected to sequencing.
reverse transcriptase / RNA polymerase or RNA ligase may be used to incorporate the at least one
the polynucleotide sequence may be any length but comprises at least ten nucleotide bases and generally at least 1 1 , 12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleotide bases.
polynucleotide sequences of at least 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 4000, 6000 or 10000 bases may be examined using the present invention.
polynucleotide sequences i.e. multiple molecules
a signalling means e.g. labels
the polynucleotide to be sequenced is a single molecule
a single polynucleotide refers to an individual molecule for sequencing by the method described herein. In this case, the data of a single molecule is used to derive the sequence. In this embodiment, where desired more than one molecule may be sequenced simultaneously using the method, but in that case each single polynucleotide's base(s) are identified separately.
the method in this embodiment relies on the use of a signalling means that produces a signal that is detectable even when only a single signalling means, e.g. label, is present, e.g. a single bead, as discussed hereinafter.
each of the polynucleotides has the same sequence to be sequenced at the same position at the 3' end of the molecule.
the multiple polynucleotides to be sequenced are identical. Means for generating populations of identical
polynucleotides is well known in the art, e.g. by the use of PCR.
the polynucleotide to be sequenced is single stranded and thus typically comprises only one DNA or RNA strand. If the sequence of a double stranded or partially double stranded polynucleotide is to be determined using the method of the invention, an initial step of melting can be carried out, e.g. by subjecting the double stranded polynucleotide to increased temperature and/or reduced salt conditions which results in the production of single stranded polynucleotides. Particularly, the polynucleotide may be subjected to temperatures of at least 70, 80 or 85°C e.g. preferably 90 °C. Such a melting step may thus be carried out in step 1 ) of the method of the invention for the provision of a single stranded polynucleotide.
the polynucleotide whose sequence is to be determined should be single stranded to allow the binding of a complementary test base(s) or test probe(s) for sequence determination, it is possible that parts or portions of the polynucleotide which are not desired to be sequenced may be double stranded. These portions will remain double stranded for the duration of the method.
the polynucleotide sequence is immobilised on a solid support, as discussed below, it is possible that its immobilised end (preferably the 5' end) is immobilised by binding to a complementary binding partner. Such binding may render the terminal part of the polynucleotide double stranded.
any part of the polynucleotide whose sequence is to be determined is single stranded.
the polynucleotide is immobilised on a solid support.
immobilised refers to direct or indirect immobilisation to a support, for example, by binding to another molecule, which is bound to the support.
Direct immobilisation may be achieved by chemical coupling and indirect immobilisation may be achieved for example by coupling through binding partners, as described hereinafter.
the "solid support” may be any solid support, for example a slide e.g. a glass slide, microarray, microparticle etc or may be an apparatus adapted for detecting a signal, e.g. for detecting a bead as described further below e.g. as identified in PCT/GB2010/00324, which is hereby incorporated by reference (i.e. a chip for optical detection or for magnetic detection).
the solid support e.g. chip, may be modified to allow appropriate binding of polynucleotides, e.g. to allow binding at specific sites to allow performance of the method and detection of a signal, e.g. a bead.
the at least one test complementary base or test probe is bound to the polynucleotide whose sequence is to be determined.
the polynucleotide sequence of the invention is thus single stranded to allow the binding of the at least one complementary test base or test probe and also to allow the binding of a recognition probe and optionally a complementary oligonucleotide sequence attached 5' to the polynucleotide portion whose sequence is to be determined, providing a primed polynucleotide sequence which can be extended e.g. by the incorporation of complementary test bases by polymerisation or by the incorporation of probes by ligation.
recognition probes are typically double stranded with single stranded protrusions which may bind to a part of the single stranded polynucleotide or may be double stranded without protrusions.
An additional complementary primer may need to be added where any step of melting is carried out after the binding of such double stranded recognition probes to the polynucleotide.
the recognition probes may be single stranded but capable of forming hairpin structures and thus of acting as double stranded molecules.
Contacting refers to bringing the polynucleotide and recognition probe and the at least one test complementary base(s) or test probe(s) into contact under conditions that allow formation of complementary base pair binding if the recognition probe, test probe or complementary test base has complementary bases to the one or more bases in the polynucleotide, or allows ligation of the recognition probe to the polynucleotide
the recognition probe may bind to the polynucleotide by hydrogen bonding or may be ligated to the polynucleotide.
Covalent binding may be achieved by any method or technique which allows the binding of a complementary test base or test probe or recognition probe to its complementary sequence in the polynucleotide sequence.
a single base i.e. a complementary test base
Such incorporation of the complementary test base or test probe will typically extend the complementary nucleotide strand in the 5' to 3' direction.
Ligation refers to the formation of a covalent bond or linkage between the terminal ends of two or more nucleic acids in a template driven reaction or a template independent reaction where the ligation may occur enzymatically or chemically. Ligation may be achieved using DNA ligase for DNA sequences e.g. T4 DNA ligase and RNA ligase for RNA sequences, e.g. T4 RNA ligase.
DNA ligase for DNA sequences e.g. T4 DNA ligase
RNA ligase for RNA sequences
the recognition probe is contacted with the polynucleotide and subsequently a ligase is added. Unligated recognition probe may then be removed, e.g. by washing.
a template driven ligation reaction refers to a ligation that is mediated by a nucleic acid template containing regions of complementarity for the ends of said nucleic acids, wherein the ends of the nucleic acid molecules to be ligated are brought into juxtaposition for ligation by hybridising to the template.
the nucleic acid template does not need to be a separate nucleic acid molecule, i.e. the template may form part of one of the nucleic acids being ligated.
the recognition probe may be ligated to the polynucleotide.
the ligation between the recognition probe and the polynucleotide may be templated by part of the recognition probe, e.g. when the recognition probe comprises a single stranded protrusion or overhang, the overhang may hybridised to the
a template independent ligation reaction refers to a ligation reaction that does not require a template nucleic acid, e.g. a ligation reaction between two non- complementary single stranded nucleic acid molecules.
Various enzymes known in the art are capable of facilitating a template independent reaction, e.g. the RNA ligase from Methanobacterium thermoautotrophicum (Mth RNA ligase).
the recognition probe may be ligated to the polynucleotide in a template independent ligation reaction.
the ligation reaction may be catalysed by a ligase capable of ligating single stranded nucleic acids in a template independent reaction.
base or “nucleotide” as used (interchangeably) herein includes the natural nucleotides of adenine, guanine, cytosine, thymine and uracil, particularly in the 2'-deoxy form or non-natural nucleotides which function in the same way, i.e. form a complementary base pair with a natural nucleotide and can be incorporated into a polynucleotide sequence by polymerisation or ligation.
complementary base refers to a base which specifically base pairs with a base to be identified in the polynucleotide.
an incorporated complementary base will be an adenine if the base to be identified in the
polynucleotide is a thymine or will be a guanine if the base to be identified in the polynucleotide is a cytosine and vice versa.
the "portion" of the test probe “which may be complementary to a region of one or more bases in said polynucleotide” refers to a sequence of one or more nucleotides or bases in the test probe which is capable of binding to the target polynucleotide when they are complementary.
the recognition probe may also comprise a portion which may be complementary to a region of one or more bases in the polynucleotide.
the portion of the recognition probe which may be complementary to a region of one or more bases in said polynucleotide refers to an overhang or protrusion of the recognition probe or a part thereof as discussed below.
the portion of the recognition probe may be complementary to a first region of one or more bases in the polynucleotide, whereas the portion of the test probe may be complementary to a second region of one or more bases in the
the recognition probe and test probe therefore bind to different regions of the polynucleotide.
the first region of one or more bases in the polynucleotide may bind a recognition probe and the second region is the binding site for the test probe, where the first and second regions are not at the same position on the polynucleotide i.e. are distinct. These regions may therefore be different lengths and/or comprise different sequences. Alternatively, the first and second regions could be the same lengths and/or comprise the same sequence but are always at different locations in the polynucleotide
test base or test probe refers to a probe or base which may exhibit the desired complementarity to the target sequence. As described hereinafter, a limited number of permutations are possible, for example for a single base only 4 permutations are possible. Test bases or test probes are used to present different permutations to establish if the base or probe has complementarity to the polynucleotide and hence will bind to that sequence. Test bases or test probes without the desired complementarity will not bind to the target sequence. Incorporated test bases or test probes refers to test bases or test probes which bind to the polynucleotide during a sequencing reaction and which are thus incorporated into the complementary nucleotide strand. Such incorporated test bases therefore are complementary to the polynucleotide at a particular position and such incorporated test probes have at least a portion which is complementary to the polynucleotide.
the improved sequencing method of the invention concerns the use of a recognition probe which allows a polynucleotide sequence to be digested or cleaved at a particular position and preferably when its
complementary nucleotide strand has been extended to a particular length.
nucleases which only digest double stranded nucleic acids
the extension of the complementary nucleotide strand results in the formation of the cleavage site by virtue of the creation of a double stranded polynucleotide.
it is still desired that the complementary nucleotide strand in the non-lagging strands is extended at least until the cleavage site.
recognition probe refers to the probe which binds to the single stranded polynucleotide sequence and which comprises a recognition site for a nuclease.
This probe is typically double stranded with a single stranded protrusion or overhang at one end or may be double stranded without a protrusion or overhang.
the recognition probe may be single stranded but capable of forming a hairpin structure and thus of acting in a similar way to double stranded DNA (such probes are referred to herein as "self-priming" as indicated previously).
the probe may bind using its single stranded protrusion or overhang to the single stranded polynucleotide sequence.
a recognition probe may bind to the end of a polynucleotide sequence (preferably the 3' end of the polynucleotide).
the probe may simply be annealed to the polynucleotide sequence by hydrogen bonding or may be ligated to the polynucleotide using a ligase.
the recognition probe must be long enough to comprise a recognition site for a nuclease and optionally an overhang that may be complementary to a portion of the polynucleotide sequence.
the recognition probe may be at least 8, 9, 10, 1 1 , 12, 13, 14 or 15 nucleotides in length e.g. from the end of the single stranded protrusion where this is present.
the probe length may be doubled for single stranded recognition probes capable of forming hairpin structures.
Such single stranded recognition probes may be at least 16, 18, 20, 22, 24, 26, 28 or 30 nucleotides in length.
the single stranded protrusion portion of a recognition probe may be at least 1 , 2, 3 or 4 nucleotides in length.
the single stranded protrusion portion is 2, 3 or 4 nucleotides in length.
the recognition probe binds to the
any overhang or protrusion of the recognition probe may be complementary to one or more bases in the polynucleotide. It is therefore possible that only a portion of the recognition probe e.g. a portion of any protrusion or overhang of the recognition probe is capable of hybridising to the polynucleotide. For example if the overhang or protrusion is 3 or 4 nucleotides in length, it is possible that only 1 , 2 or 3 nucleotides of such protrusions are capable of binding to the polynucleotide sequence e.g. are complementary to the polynucleotide.
any part or portion of the recognition probe may allow the binding.
the full overhang of the recognition probe is capable of binding to said polynucleotide such that the recognition probe and said polynucleotide are bound without a gap, i.e. each strand of the double stranded molecule that results from the binding is continuous and there is no requirement to fill in the sequence, e.g. during ligation.
binding may be achieved by any appropriate means, e.g. by applying moderate heat and washing after ligation.
repeated cycles of ligation and washing may be performed to maximize binding.
Polynucleotides to which no recognition probe is bound may be effectively removed from further cycles, e.g. by appropriate chemical or enzymatic methods, such as blocking the 3' ends to prevent ligation of a recognition probe in a later cycle. Such techniques are well known in the art, e.g. as described herein.
the probes may be removed from further cycles by degradation, e.g. enzymatic degradation, such as exonuclease digestion.
Suitable exonuclease enzymes include Exonuclease I or Exonuclease T, (from E. coli).
blocking and/or digestion is performed after step 4), i.e.
the recognition probe preferably binds, as described above, such that the recognition probe and the polynucleotide are bound without a gap.
Protocols in which only part of the overhang of the recognition probe is involved in hybridising to the polynucleotide may be used when the method is performed on multiple molecules, but in that case the recognition probe must bind in the same position in each case, i.e. generating the same gap on each resultant double stranded polynucleotide.
the recognition probe may additionally comprise universal nucleotides.
Universal nucleotides refer to nucleotides that exhibit the ability to replace any of the four natural nucleotides without significantly destabilising neighbouring nucleotides, for example 3-nitropyrole 2'-deoxynucleoside and 5-nitroindole 2'- deoxynucleoside.
any protrusion or overhang of the recognition probe comprises one or more universal nucleotides which will be capable of hybridising with any of the 4 natural nucleotides occurring in the polynucleotide (or non-natural nucleotides).
the use of one or more universal nucleotides in the recognition probe protrusion may therefore reduce the number of different recognition probe variants that may be required to be used in the present invention in a recognition probe set (discussed in detail below).
a universal nucleotide is used in a particular position in any protrusion, such a nucleotide will bind to any of A, T/U, C, G in the polynucleotide and thus there is no need to create probes with any variation at that position (as would usually be necessary to ensure at least one recognition probe is present that is complementary to the
the protrusion or overhang of the recognition probe may solely consist of universal nucleotides.
Such a recognition probe may be capable of binding to any single stranded polynucleotide.
a spacer may be present in the recognition probe e.g. between the nuclease recognition site in the recognition probe and any single stranded protrusion.
any such spacer will be small e.g. less than 4, 3 or 2 nucleotides in view of the cleavage range of the enzyme from its recognition site. If too large a spacer is present, the sequencing reaction will only generate a short complementary nucleotide strand before a cleavage/digestion site is formed.
a recognition probe set is contacted with the polynucleotide sequence.
reference to a recognition probe includes reference to a recognition probe set.
Such a probe set preferably will comprise recognition probes having different or varied overhangs from one another when such recognition probes are being used.
the overhangs will be designed so that one of the probes present in a probe set will be capable of binding to the
the probe set may comprise probes having protrusions of the sequence AA, TT, CC, GG, AT, TA, CG, GC, AG, GA, CA, AC, TC, TG, CT and GT i.e. all possible permutations of A, T, C and G (T may be substituted with U for RNA sequences).
the probe set may comprise probes having protrusions of the sequence; AAA, TTT, CCC, GGG, ATT, ACC, AGG, ATA, ACA, AGA, TAA, TGG, TCC, TAT, TGT, TCT, CTT, CGG, CAA, CTC, CGC, CAC, GAA, GTT, GCC, GAG, GTG, GCG etc.
recognition probes may comprise one or more universal nucleotides e.g. in the protrusion which may reduce the number of variants required to be used in a sequencing cycle.
the recognition probe comprises a recognition site for a nuclease, preferably for a type II or type III restriction enzyme.
the recognition site is for any one of the following type II restriction enzymes; Alol, Arsl, Bael, Barl, Bpil, Bsp241 , Fall, Hin4l, NmeDI, Ppil, Psrl, Tstl, Aarl, Acc36l, Acelll, Bbsl, BfuAI, BtgZI, Eco31 l, Eco0441 , Espl, Fokl, Lwel or is for a type III restriction enzyme such as EcoP15l.
the recognition probe preferably comprises a single nuclease recognition site but it may comprise more than one recognition site for more than one nuclease.
some nucleases e.g. some type III restriction enzymes require the presence of more than one recognition site to function e.g. two recognition sites.
the recognition probes of the invention may therefore also comprise more than one recognition site for a single enzyme, depending on the enzyme to be used in the cleavage step of the method.
a nuclease with a long cutting reach is preferred in the invention i.e. an enzyme that cleaves a polynucleotide sequence as far from its recognition site as possible.
the enzymes used in the invention may typically cleave or digest the polynucleotide at least 10, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24 or 25 bases from the recognition site.
a preferred nuclease to be used in the method of the invention in this respect is EcoP15l, which is a type III restriction enzyme. EcoP15l cleaves 25 to 27 bases away from its recognition site and thus has a long reach.
the nuclease to be used in the present invention should preferably have an accurate distance cut and should hence cleave each polynucleotide at a specific distance from the recognition site. This is particularly important for multimolecule sequencing reactions to ensure the synchronisation of all strands.
the enzyme used should accurately cut all strands at the same position i.e. the same distance from the recognition site. Preferably at least 90, 95, 99 or 100% of polynucleotide strands should be cut at the same distance from the recognition site in the recognition probe.
an enzyme for use in single molecule sequencing may not need to have an accurate cut distance e.g. the cleavage distance could vary by at least 1 , 2, 3, 4, or 5 nucleotides between the cleavage reactions used after each sequencing cycle.
the cleavage distance could vary by at least 1 , 2, 3, 4, or 5 nucleotides between the cleavage reactions used after each sequencing cycle.
an enzyme could cleave the polynucleotide at a position which is x nucleotides from the recognition site in a first sequencing cycle and at a position which is x+/- 1 in a second sequencing cycle (or x+/- 2, 3, 4 or 5).
nucleases may be used in the invention which cleave only double stranded nucleic acids or which cleave double stranded and single stranded nucleic acids.
Nucleases which only cleave double stranded nucleic acids may cleave less than 10, 8, 5, 4, 3, 2 or 1 % of single stranded molecules. Thus preferably, at least 90, 95, 96, 97, 98 , 99% of nucleic acid molecules cleaved by nucleases which only cleave double stranded nucleic acids will be double stranded.
a nuclease can be used which only cleaves single stranded nucleic acids, this is not preferred and in this embodiment, the complementary nucleotide strand would not be extended to the cleavage site.
the nuclease is capable of cleaving only double stranded nucleic acid molecules or is capable of cleaving both double stranded and single stranded nucleic acid molecules.
the recognition probe may have cross-linked strands to prevent its removal from the polynucleotide during any desired step of melting as discussed previously.
crosslinking can be achieved for example by using alkylating agents, platinum compounds and psoralen or by using any other compounds/methods known in the art e.g. as discussed in Noll et al, Chem. Rev., 2006, 106(2), 277-301 , which is incorporated herein by reference.
the recognition probe may comprise, be conjugated to or have a signalling means attached.
signalling means are discussed below, but particularly include a bead e.g. a magnetic bead.
the signalling means may allow for confirmation that a recognition probe has bound to a polynucleotide and/or may allow unbound recognition probe to be removed from the reaction.
a recognition probe will bind to the polynucleotide sequence during contact step 2) of the method.
the recognition probe may be added either before, at the same time or sequentially to the complementary test bases/test probes. If the recognition probe is added after the test bases/test probes, the identification of the nucleotide sequence in the polynucleotide will not occur until the recognition probe has bound to the polynucleotide and the polymerase/ligation reaction has begun.
Step 4) of the method of the invention allows one or more nucleotides in the polynucleotide to be determined.
the identification of one or more nucleotides in the polynucleotide allows one or more nucleotides in the polynucleotide to be determined.
nucleotides in the polynucleotide is determined by identifying which complementary test base or test probe has been incorporated into the generated complementary nucleotide strand at a particular position i.e. in step 3) by detecting a signal e.g. generated by the incorporation (e.g. Pi released during polymerisation) or a signal on a signalling means conjugated or attached to that complementary test base or test probe.
sequencing methods such as sequencing by synthesis, sequencing by ligation, Pyrosequencing or Ion semiconductor sequencing may be employed.
the primed polynucleotide with all of the possible complementary test bases of A, T/U, C and G in one reaction if they are associated with different signalling means and to determine which complementary test base is incorporated by detecting the signalling means attached to that complementary test base.
the complementary test bases may each be conjugated or attached to a different signalling means in order to determine which has been incorporated.
all complementary test bases be conjugated to the same signalling means although different signalling means can be used. In this instance, the absence of a signal from the polynucleotide after contact with a complementary test base indicates that the base has not been incorporated.
test probe refers to a single stranded probe which is at least 2 nucleotides in length and which may have a portion complementary to the polynucleotide.
a set of test probes is contacted with the polynucleotide where such test probes encompass all or most possible sequence variant test probes of a particular length. Reference to a test probe therefore encompasses a set of test probes. It is not necessary for all the nucleotides within a test probe to be defined.
test probes may be contacted with the polynucleotide which have at least one known base in a particular position.
the known nucleotide may be at any position in the test probe.
test probes may be generated with the sequences ANNN, TNNN, CNNN, GNNN where N may be any other nucleotide.
a probe set may be produced encompassing all possible permutations of each different nucleotide as N.
Each probe type e.g.
ANNN, GNNN, TNNN, CNNN may be conjugated to a different signalling means if the probes are to be added to the polynucleotide at the same time or alternatively each probe type may be conjugated to an identical signalling means if they are to be contacted with the polynucleotide separately.
the detection of a nucleotide is possible by detecting which signalling means is present and hence which probe type has been incorporated.
the detection of a nucleotide is possible by detecting the presence of a signalling means. The absence of a signalling means indicates that the test probe has not been incorporated and thus that the nucleotide in the probe is not complementary to the polynucleotide.
Test probes of different lengths are possible e.g. of at least 2, 3, 4, 5, 6, 7, or 8 nucleotides and thus probes of the form ANx, TNx, CNx and GNx may be used where N is any nucleotide and x is at least 1 , 2, 3, 4, 5, 6 or 7.
the probes may comprise at least one known nucleotide at any position in the probe.
probes of the form NANN, NNAN or NNNA could be used where the known nucleotide can be at any defined position in the probe.
more than one nucleotide may be known in a probe e.g. 2, 3, 4 or more nucleotides may be known at particular positions.
the known nucleotides may be consecutive or non-consecutive in a probe.
a test probe may further comprise one or more universal nucleotides in addition to the one or more known nucleotides.
a polynucleotide which is sequenced using test probes where not all the nucleotides are defined may need to be subjected to several rounds of sequencing to achieve a full polynucleotide sequence. Thus, it may not be possible to determine all of the required nucleotides by generating a complete
the probes used are only capable of identifying for example, one or two nucleotides in their length.
test probes that comprise 2 or more known nucleotides may be used to determine the sequence of two or more nucleotides in the polynucleotide during a ligation reaction.
Step 3) in the method of the invention hence requires that at least one complementary test base/test probe is contacted with the polynucleotide.
said at least one complementary test base/test probe will bind to the polynucleotide when said at least one complementary test base or said portion of at least one test probe is complementary to one or more bases in the polynucleotide.
Step 3) can be repeated as many times as necessary in order that at least one complementary test base/test probe has bound to the polynucleotide.
step 3) may need to be repeated multiple times until binding of at least one complementary test base/test probe is achieved.
step 3) can be carried out one or more times to bind more than one complementary test base to the polynucleotide, before any step 4) of identification is carried out. This may occur if the complementary test base does not terminate the reaction.
test base or test probe may have a terminating effect preventing further extension or incorporation of other bases or probes into the sequence.
complementary base or probe is terminating on the binding of further
probes which are added for ligation methods these probes essentially terminate the reaction (e.g. until the cleavage reaction takes place) and thus no terminating nucleotides are required on said probes though terminating bases may be used, e.g. in which the 5' is dephosphorylated.
terminating bases may be used, e.g. in which the 5' is dephosphorylated.
the 3' end of the probes may be phosphorylated to prevent unwanted ligation, wherein the phosphorylation can be removed after the ligation reaction with, e.g. a
step 4 i.e. identifying which complementary test base or test probe has been incorporated into the generated complementary nucleotide strand at a particular position, e.g. using sequencing methods, before the probes are blocked to prevent unwanted ligations.
Binding of the complementary probe or base may have a terminating effect if a large signalling means, e.g. a bead, is selected such that its size, e.g. radius is larger than the length of the target polynucleotide. In that case a second signalling means carrying a probe or base is unable to access the target polynucleotide essentially terminating any further extension or binding. In this way even homopolymers may be identified correctly, provided that if multiple bases or probes are carried on a single carrier which acts as a label, e.g. a bead, the bases on the carrier are placed so that two consecutive bases cannot be incorporated on the same target from the same carrier.
An advantage with such a method is that there is no need for fluid exchange, or the fluid may be reused directly.
a signalling means may be associated with said test probe(s) or test complementary base(s).
a combination of unmodified and modified test probes or test bases may be used in the method of the invention e.g. where only the identification of particular nucleotides is required or where it is desired to extend the complementary nucleotide strand further than the cleavage site e.g. when using a nuclease which only cleaves double stranded nucleic acids.
a “signalling means” is any moiety capable of direct or indirect detection by the generation or presence of a signal. The term “signalling means” or “labelling means” or “label” are used
a "signal” as referred to herein may be any detectable physical characteristic such as conferred by radiation emission, scattering or absorption properties, magnetic properties, or other physical properties such as charge, size or binding properties of existing molecules (e.g. labels) or molecules which may be generated (e.g. gas emission, release of Pi etc.). Techniques may be used which allow signal amplification, e.g. which produce multiple signal events from a single active binding site, e.g. by the catalytic action of enzymes to produce multiple detectable products.
the signalling means may be a labelling means (e.g. a label) which itself provides a detectable signal or a means for generating a detectable signal. Conveniently this may be achieved by the use of a radioactive or other label which may be incorporated during probe production or conjugated directly to bases or probes to be used in the invention.
a labelling means e.g. a label
this may be achieved by the use of a radioactive or other label which may be incorporated during probe production or conjugated directly to bases or probes to be used in the invention.
the signal is provided by a labelling means associated with said recognition probe, test probe or test complementary base, which is preferably attached to said recognition probe, test probe or test
label are those which directly or indirectly allow detection or measurement of the binding of the recognition probe, test probe(s) or test base(s) to the target polynucleotide.
labels include for example radiolabels, chemical labels, for example chromophores or fluorophores (e.g. dyes such as fluorescein and rhodamine), or reagents of high electron density such as ferritin, haemocyanin or colloidal gold.
the label to be used is not a bead. However, particularly for single molecule sequencing the label may be a bead.
the label may be an enzyme, for example peroxidase or alkaline phosphatase, wherein the presence of the enzyme is visualized by its interaction with a suitable entity, for example a substrate.
the label may also form part of a signalling pair wherein the other member of the pair is found on, or in close proximity to, the probe or base which binds to the target polynucleotide, for example, a fluorescent compound and a quench fluorescent substrate may be used on adjacent binding bases and probes.
the signalling means may be associated with the recognition probe, test probe or test base indirectly e.g. through binding partners one of which carries a signalling means as discussed hereinafter.
a first binding partner of a binding pair is attached to said recognition probe, test probe or test complementary base and said signalling means is attached to a second binding partner of said binding pair and said first binding partner is attached to said second binding partner during said method.
a label may be provided on a different entity, such as an antibody, which recognizes a peptide moiety attached to the recognition probe, test probe or test base, for example attached to a base used during synthesis of the test probe.
the signalling means may be associated with the recognition probe, test probe or test base at any convenient point during the process.
step 3) is performed by determining the presence or absence of a signal associated with the test base or test probe, where the presence of signal is indicative of binding of the test base or test probe.
the labelling means may be a bead particularly for single molecule sequencing e.g. bead labelled probes and/or complementary test bases may be used.
the "signal" of said bead is the signal which is detected, e.g. its magnetism, optical activity, fluorescence, colour and so on, depending on the nature of the bead used.
the signalling means as well as their linking groups to the test base or test probe for each permutation may be different and may be distinguishable allowing their addition together and separate detection during the method.
the signalling means for all test probes may be identical and each test probe set may be added sequentially or separately until the test probe set with the relevant complementary probe or base is employed. There is no need to determine which recognition probe has bound to the polynucleotide and thus there is no requirement to use different signalling means or linking groups for different recognition probes. However, this can be used if desired.
a complementary test base, test probe or recognition probe may be either labelled with a signalling means prior to binding to the polynucleotide or may be labelled with a signalling means after binding to the polynucleotide through the use of binding partners. If labelled after binding, a step of removing any unbound signalling means should be carried out prior to any step of determining the presence of a signalling means attached to the bound test base, test probe or recognition probe.
test base/test probe/recognition probe is labelled prior to binding
a step may be carried out to remove any unbound test base/test probe/recognition probe signalling means labelled complexes before the step of determining the presence of a signalling means attached to the bound test base, test probe or recognition probe.
the labelled probe or base may be detected in situ (i.e. when attached to the target sequence) or detected on release. In the latter case, the sequencing reaction is washed to remove unbound signalling means from the mixture and then the signalling means are released as described hereinbefore by cleavage. The presence of signalling means in the released mix is evidence of the presence of the corresponding complementary base(s) in the target sequence.
signalling means Before assessment of the next set of test probes or bases may commence the signalling means must be removed from the polynucleotide (or from released probes or bases for reuse). In methods in which signalling means are detected in situ, after their detection they must be released (by cleavage) and removed.
signalling means may be released by an enzymatic cleavage step.
the signalling means may be released by chemical cleavage.
a cleavage site may be placed between the base or probe and the signalling means, where any cleavage may be achieved
a restriction enzyme site may be incorporated between the signalling means and the base or probe. Any restriction enzyme may be used and cleavage may then be achieved using any suitable restriction enzyme for that site. However, preferably a different restriction enzyme is used to release the signalling means from the restriction enzyme that recognises the recognition site in the recognition probe.
beads are used as the signalling means e.g. for single molecule sequencing, they are preferably magnetic allowing ease of removal, for example using a magnet if the beads are paramagnetic.
beads refers to a microparticle which is typically but not necessarily a spherical solid support.
the size of the beads is not critical, they may for example be of the order of diameter of at least 0.05, 0.1 , 0.3, 0.5, 1 , 1 .5, 2, 2.5, 3 or 3.5 ⁇ and have a maximum diameter of not more than 50, 20, 10, 8 or 6 ⁇ . Particularly, beads of 1 or 2.8 or 4.5 or 10 ⁇ may be used in the invention.
the signalling means may be attached to the test complementary test base or test probe either directly or indirectly in any convenient way before or after incorporation into the polynucleotide or to the recognition probe before or after it is bound to the polynucleotide, according to techniques well known in the art and described in the literature but ensuring that the signalling means if it is large, e.g. a bead, does not prevent access of the probe or base to which it is attached to the target polynucleotide or prevent required reactions to take place, e.g.
test base, test probe or recognition probe may be attached directly to the signalling means.
attachment may readily be achieved by methods (e.g. coupling chemistries) well known in the art and conveniently, the base or probe may be bound directly to the signalling means.
the signalling means is large, more than one base or probe may be attached such that the signalling means acts as a carrier, e.g. the test base, test probe or recognition probe may be used to coat the carrier.
the signalling means may be indirectly attached to the test complementary test base or test probe or recognition probe.
the test base or test probe or recognition probe may therefore be attached to the signalling means through one or more other molecules which may be directly attached to the signalling means. These may give rise to a covalent or non-covalent association.
the signalling means may carry one or more linking moieties or spacers which have an affinity for the test base, test probe or recognition probe or for a tag incorporated into the test base, test probe or recognition probe.
the signalling means may conveniently carry or be provided with a binding moiety capable of binding to the test base, test probe or recognition probe such that binding occurs via at least two binding partners of a binding pair.
a binding pair refers to a pair of molecules which form a specific and stable interaction. Examples included DNA:DNA, ligand:receptor, antibody:antigen interactions.
binding moieties are well known in the art e.g. biotin/streptavidin may be used where the test base, test probe or recognition probe is coupled to a biotin group and if beads are used as signalling means these may be streptavidin coated.
test base test probe or recognition probe may be attached to the signalling means by biotin/streptavidin binding or by biotin/avidin binding in which biotin and streptavidin form the binding partners.
streptavidin or avidin coated signalling means may be used to bind a test base, test probe or recognition probe which is linked to a biotin group.
Other binding pairs which may be used include digoxigenin:antidigoxigenin.
the test base or test probe or recognition probe is attached to said signalling means via a linkage (preferably but not necessarily including binding pairs), which is cleavable e.g. by chemical cleavage or by enzymatic cleavage.
a linkage preferably but not necessarily including binding pairs
said cleavable linkage may have a restriction site cleavable by a restriction enzyme or may be cleaved chemically. Conveniently this may be generated by use of at least partially single stranded oligonucleotides which are binding partners which together form a recognition and restriction site once hybridized.
test base or test probe or recognition probe may be attached to the signalling means prior to binding to the polynucleotide.
test base or test probe or recognition probe may first be bound to the polynucleotide sequence before being attached to the signalling means.
the signalling means may conveniently carry or be provided with one of a pair of binding partners as described hereinbefore.
Coloured transparent beads can be used in the methods of the invention as the signalling means and can be detected.
the different sets of test probes or nucleotides (bases) to be tested may be added together or separately depending on the detection technique to be used.
the detection of the signalling means may, depending on the signalling means, be by optical, magnetic, electric or electrochemical means.
Many detectors/instruments are known and available in the art for detecting signals and thus an incorporated complementary test base and/or test probe e.g. such instruments are available from lllumina.
an optical detection method using a chip may be employed as described in
step 4) of the invention of identifying one or more nucleotides in the polynucleotide can be carried out by detecting a wide variety of signalling means.
Step 5) of the invention involves optionally repeating steps 3) and/or 4) one or more times.
complementary test bases may be used in step 3) which have a terminating effect on the complementary nucleotide strand extension, once incorporated.
a step 4) of identification may be carried out after each individual complementary base has bound. In this way, multiple complementary test bases are incorporated and their corresponding nucleotides in the polynucleotide determined before any step 6) of cleavage is carried out.
step 5) is optional, it is preferred that this step be carried out. In cases where step 5) i.e. repeating steps 3) and/or 4) is not carried out, at least 2 complementary test bases/probes should be bound to the polynucleotide in step 3). In this instance, it may be desirable to identify the at least two complementary test bases/probes separately e.g. by using different signalling means.
steps 3) and/or 4) are preferably repeated at least 2 or more times, e.g. at least 3, 4, 5, 6, 7, 8, 9, 10, 15 or 20 times.
the complementary nucleotide strand is of a length which may be cleaved by a nuclease recognising the recognition site in the recognition probe, the polynucleotide may be cleaved with the nuclease.
cleaved refers to the complete cleavage or digestion of the polynucleotide and the complementary nucleotide strand (i.e. of the double stranded entity formed by the extension of the complementary nucleotide strand on the polynucleotide) or to the complete cleavage or digestion of the polynucleotide at a single stranded site downstream of the bound complementary nucleotide strand.
Downstream of the bound complementary nucleotide strand means at least 1 , 2, 3, 4 or 5 nucleotides downstream of the last incorporated complementary test base or test probe in the complementary nucleotide strand.
any single stranded cleavage is generally carried out for lagging polynucleotide strands and not non-lagging polynucleotide strands).
Cleavage occurs at a specific distance from the nuclease recognition site in the recognition probe bound to the polynucleotide as discussed previously and is carried out by a nuclease as defined herein.
the nuclease used in the present invention preferably either has only the ability to cleave double stranded nucleic acids or has the ability to cleave both double stranded and single stranded nucleic acids.
the site of cleavage is generated by the generation of the complementary nucleotide strand.
the complementary nucleotide strand it is necessary in this aspect for the complementary nucleotide strand to be present at the required distance from the nuclease recognition site in order for cleavage or digestion to occur. In this way, it is necessary in this aspect for the complementary nucleotide strand to be extended or generated to at least the minimum length required for cleavage by the nuclease, e.g. to the reach of the nuclease enzyme (i.e. to the point that the enzyme would cleave) or beyond if the enzyme requires the presence of additional residues beyond the cleavage site to be effective e.g. at least 1 , 2, 3 or 4 residues beyond the cleavage site of the enzyme.
the complementary nucleotide strand in order for cleavage of the polynucleotide and the complementary nucleotide strand to occur when using a nuclease which only cleaves double stranded nucleic acids, the complementary nucleotide strand must have been extended to a sufficient length for the generation of a cleavage site.
the complementary nucleotide strand is extended to a length of at least x nucleotides, preferably to a length of x+1 , x+2, x+3, x+4, x+5, x+6, x+7, x+8, x+9, x+10, x+15 or x+20 nucleotides before cleavage of the polynucleotide and the complementary nucleotide strand is effected.
cleavage can be carried out once a particular portion of the polynucleotide sequence has been determined or alternatively, cleavage can be carried out once a specific time period has passed allowing for the
complementary nucleotide strands to reach a certain length.
the complementary nucleotide strands are extended at least 1 or 2 nucleotides beyond the cleavage site of the enzyme. This may ensure that all polynucleotide and complementary nucleotide strand duplexes are capable of being cleaved particularly when using a nuclease which only cleaves double stranded nucleic acids.
both non-lagging and lagging strands have complementary nucleotide strands bound which are sufficiently extended to allow cleavage.
the site of cleavage may not necessarily be generated by the generation of the complementary nucleotide strand for all polynucleotides in the reaction.
the non-lagging strands i.e. the strands which are synchronised and have been sequenced up to (and/or including) the cleavage site, may be double stranded at the cleavage site i.e. the complementary nucleotide strand is extended at least to the cleavage site.
any lagging strands i.e. strands which are 1 , 2, 3 or more bases behind the sequencing reaction of the non-lagging strands, may still be single-stranded at the cleavage site, as the complementary nucleotide strand may not yet have extended this far.
the nuclease may cleave both the polynucleotides which are double stranded and any which are single stranded.
the non-lagging strands may be double stranded at the cleavage site and the lagging strands may also be double stranded if the complementary nucleotide strand is extended past the cleavage site.
both types of cleavage may occur.
the complementary nucleotide strand is extended or generated to at least the cleavage site e.g. to the reach of the nuclease enzyme, but it may not be necessary to extend the complementary nucleotide strand beyond that point (although this can of course be done as described previously for nucleases which only cleave double stranded nucleic acids).
test bases or test probes when using a nuclease which only cleaves double stranded nucleic acids, it is possible to add unlabelled or unmodified test bases or test probes after the desired portion of polynucleotide sequence has been determined. Such unlabelled bases/probes may be more easily incorporated into the
complementary nucleotide strands are extended to the cleavage site.
Such a step can also be carried out when using a nuclease which cleaves both single and double stranded nucleic acids, although this may not be necessary.
a further step may take place after method step 4) or step 5) when it is performed but before cleavage step 6) of incorporating unmodified bases or probes into the complementary nucleotide strand to extend its length to ensure cleavage in step 6) by a nuclease. It is possible for sequencing reactions which have been carried out using modified probes and ligation that this further method step could be carried out using either unmodified bases with polymerase or unmodified probes and ligation.
an alternative polymerase enzyme to the polymerase used for the incorporation of modified bases
Such an alternative polymerase may be more efficient than the polymerase used in the sequencing reaction and hence may assist in ensuring the complementary nucleotide strands are extended to the cleavage site.
the cleavage enzyme may be contacted with the polynucleotide and its complementary nucleotide strand after a sequencing cycle has been carried out and the desired length of complementary nucleotide strand has been reached e.g. particularly for the synchronised strands, or alternatively, the enzyme may be present in the reaction or added to the reaction before the desired length of complementary nucleotide strand is obtained.
the nuclease can be added to the reaction at any time during or after sequencing, it is preferred that the nuclease is added after the reaction i.e. once the desired length of
nuclease which can cleave single stranded nucleic acids e.g. one which can cleave both double stranded and single stranded nucleic acids
the nuclease should only be added to the reaction once the complementary nucleotide strands have been extended to the desired length on the non-lagging strands and thus when the required bases have been sequenced in that cycle. If the nuclease is added before this point, it will cleave the polynucleotides at the single stranded sites before the required sequencing reaction is completed.
strands which are synchronised will generate the double stranded cleavage site before lagging polynucleotides and thus may be cleaved before lagging strands when the nuclease is present throughout the reaction.
the polynucleotides are all cleaved or subjected to cleavage at the same time.
complementary nucleotide strands are cleaved during the cleavage reaction for a multimolecule sequencing method.
at least 70, 80, 90, 95 or 99% of polynucleotide and complementary nucleotide strands may be cleaved during the cleavage step. (If a single molecule is being sequenced, then of course this polynucleotide should be cleaved during the cleavage reaction and the above % figures do not apply).
a further step may also be employed to prevent non-digested strands from providing a signal in any further sequencing cycle.
Irreversibly terminated bases include dideoxynucleotides.
the cleavage step of the invention preferably results in at least 70, 80, 90, 95, or 99% of the polynucleotide strands terminating at the same 3' position in the sequence and/or being the same length. If the starting material is of uniform size the strands will be the same length. Most preferably, 100% of polynucleotide strands are the same length after cleavage. In this way, the cleavage step is able to synchronise the sequencing reaction to efficiently remove or disable lagging or read-ahead strands. Further, preferably a stalled reaction may be able to be restarted e.g. where the reaction has stalled after the cleavage site.
Preferred nuclease enzymes for use in the cleavage step are discussed above, but particularly include Type II or Type III restriction enzymes. It is generally preferred that the enzyme for the cleavage step requires the presence of only one recognition site in the recognition probe to act, although enzymes that require more than one recognition site may be used (e.g. those that need 2 or 3 sites). In these cases, the recognition probe may have more than one recognition site. Further, more than one nuclease could be used in the invention although again, this would require the recognition probe to have an equivalent number of recognition sites.
the invention involves the performance of steps 2) to 4) and 6) (and optionally step 5)) of the method one or more times until the polynucleotide sequence is determined.
a further sequencing cycle can be carried out where the cleaved polynucleotides are again contacted with a recognition probe and at least one complementary test base or test probe to identify one or more subsequent nucleotides in the polynucleotide.
the number of repetitions of steps 2) to 4) and 6) (and optionally step 5)) that are carried out will primarily depend on the length of sequence that is to be determined in the polynucleotide, and the reach of the nuclease enzyme being used.
steps 2) to 4) and 6) may be carried out only once or several repetitions may be necessary e.g. at least 2, 3, 4, 10, 20, 30, 100 repetitions.
the repetition of steps 2) to 6) may further include other steps such as the melting steps described above.
the last sequencing cycle to be carried out in the method may not comprise a cleavage step 6).
the method of the invention involves repeating steps 2) to 6) e.g. one or more times, the last repeat cycle may not comprise step 6).
the polynucleotide sequence has been determined, there is no requirement to synchronise strands for another sequencing cycle.
the present invention also provides a method for achieving synchronisation and/or for preventing stalling of a sequencing reaction, wherein the method comprises the steps of:
identifying one or more nucleotides in the polynucleotide by determining which at least one complementary test base or which at least one test probe bound to the polynucleotide sequence in step 3);
nuclease which recognises the recognition site in the recognition probe bound to said polynucleotide, wherein the nuclease cleaves the polynucleotide at a site which is double stranded from the binding of said complementary test bases or said test probes to the polynucleotide sequence during step 3) or at a single stranded site on the polynucleotide which is downstream of said bound complementary test bases or said test probes,
each cycle of steps 2) to 6) is carried out one or more times until the polynucleotide sequence has been determined and wherein when a
step 3) may precede step 2).
the term "achieving synchronisation” means that typically, at least 70, 80, 90, 95 or 99% of polynucleotide strands are synchronised after the cleavage step of the method.
"Synchronised” means having the same 3' (or 5') end sequence for sequencing and/or the same length.
100% of the polynucleotide strands are synchronised after the cleavage step.
synchronised polynucleotides have been cleaved at a common site i.e. at a specific distance from the recognition site in the recognition probe and are preferably of the same length.
Polynucleotides which are synchronised during a sequencing reaction are those which are at the correct point in that sequencing reaction i.e. which have incorporated the relevant base/probe at each step and are not therefore lagging or read-ahead strands.
the degree of synchronisation in a sequencing reaction can be determined by the amount of background noise detectable i.e. any deviation in sequence readout from lagging or read-ahead strands in the sequencing reaction. Noise from desynchronisation can result in a highly predictable signal since generally this creates a signal which is one base or reaction behind the sequence signal from the synchronised strands.
the methods of the invention are designed to achieve synchronisation and thus the level of noise from
desynchronised strands should be low with the % of desynchronised strands being typically less than 30, 20, 10, 5 or 1 %.
the method of the invention provides an advantageous adaptation to highly used realtime sequencing methods of the art that employ modified nucleotides or probes.
the method prevent fading of the signal and allows the synchronisation of polynucleotides in a sequencing reaction.
Figure 1 shows the effect of lagging.
the first sequencing reaction shows the incorporation of dATP into the complementary nucleotide strand for the first two strands shown.
the bottom strand however has no incorporation of dATP.
the second sequencing reaction then proceeds where dGTP is incorporated into the top two strands (i.e. the next base along) but the bottom lagging strand incorporates dATP and hence will provide an incorrect signal.
Figure 2 shows one method of the invention, where a recognition probe is bound to a polynucleotide attached to a surface.
the recognition site in the recognition probe is shown by dashing.
the recognition probe protruding strand is then extended and is subsequently digested with nuclease once a cleavage site is generated.
a portion of the complementary nucleotide strand remains on the polynucleotide after cleavage. This is melted away leaving a single stranded polynucleotide which can be subjected to a further sequencing cycle.
Figure 3 shows one method of the invention, where a recognition probe (shown on the right of the figure) is bound, via a single stranded ligation reaction (i.e. a template independent ligation reaction) to a polynucleotide attached to a surface (shown on the left of the figure). All probes are depicted in the orientation 5' to 3' (except the primer in step 5, which is 3' to 5').
the recognition site for e.g. a type NS-enzyme, in the recognition probe is the light grey area at the 5' end of the probe.
the medium shade of grey at the 3' end of the polynucleotide attached to the surface depicts the nucleotides that will be removed.
the polynucleotide may be protected from the nuclease enzymes by methylation.
the recognition probe is preadenylated at the 5' end and phosphorylated at the 3' end.
the recognition probe is contacted with, and ligated to, the polynucleotide in steps 1 and 2.
Step 3 depicts the undesirable ligation products that may form in subsequent rounds of ligation if the 3' end of the recognition probe is not phosphorylated.
the undesirable ligation products may be removed, e.g. using an endonuclease that cleaves at a site found in the junction between two recognition probe concatamers.
the remaining unligated or uncut probes are removed in step 4 using an Exonuclease, e.g.
Exonuclease I A primer (shown as an arrow, wherein the arrow head represents the 3' end) and polymerase are added in step 5, wherein the primer is extended beyond the restriction enzyme cutting site to form the complementary strand.
step 6 the polynucleotide is cleaved and the cleaved nucleic acid is liberated by heating or using a 5' exonuclease e.g. lambda exonuclease.
the 3' phosphate is removed on uncut molecules using a polynucleotide kinase, e.g. T4 polynucleotide kinase.
step 7 the procedure is repeated until a sufficient portion of the polynucleotide is removed and the process terminates at the end of step 4.

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PCT/EP2013/068038 2012-08-30 2013-08-30 Méthode de séquençage pour polynucléotide monobrin utilisant une sonde avec site de reconnaissance pour nucléase Ceased WO2014033285A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number	Priority date	Publication date	Assignee	Title
WO2016020292A1 (fr) *	2014-08-06	2016-02-11	Geneseque As	Procédé de séquençage de polynucléotides immobilisés

Citations (7)

* Cited by examiner, † Cited by third party
Publication number	Priority date	Publication date	Assignee	Title
WO1995027080A2 (fr) *	1994-04-04	1995-10-12	Lynx Therapeutics Inc	Sequençage d'adn par ligature et clivage par etapes
WO1996037630A1 (fr) *	1995-05-22	1996-11-28	Sri International	Determination de la taille d'oligonucleotides a l'aide d'amorces scindables
US20080269068A1 (en) *	2007-02-06	2008-10-30	President And Fellows Of Harvard College	Multiplex decoding of sequence tags in barcodes
US20100009354A1 (en) *	2006-02-20	2010-01-14	National University Corporation Hokkaido University	Method for determining base sequence of dna
US20100035249A1 (en) *	2008-08-05	2010-02-11	Kabushiki Kaisha Dnaform	Rna sequencing and analysis using solid support
WO2010053820A1 (fr) *	2008-10-29	2010-05-14	Trustees Of Boston University	Conversion d'adn avec conservation de séquence
WO2012042052A1 (fr) *	2010-09-30	2012-04-05	Geneseque As	Procédé de séquençage

2012
- 2012-08-30 GB GB201215435A patent/GB201215435D0/en not_active Ceased
2013
- 2013-08-30 WO PCT/EP2013/068038 patent/WO2014033285A1/fr not_active Ceased

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number	Priority date	Publication date	Assignee	Title
WO1995027080A2 (fr) *	1994-04-04	1995-10-12	Lynx Therapeutics Inc	Sequençage d'adn par ligature et clivage par etapes
WO1996037630A1 (fr) *	1995-05-22	1996-11-28	Sri International	Determination de la taille d'oligonucleotides a l'aide d'amorces scindables
US20100009354A1 (en) *	2006-02-20	2010-01-14	National University Corporation Hokkaido University	Method for determining base sequence of dna
US20080269068A1 (en) *	2007-02-06	2008-10-30	President And Fellows Of Harvard College	Multiplex decoding of sequence tags in barcodes
US20100035249A1 (en) *	2008-08-05	2010-02-11	Kabushiki Kaisha Dnaform	Rna sequencing and analysis using solid support
WO2010053820A1 (fr) *	2008-10-29	2010-05-14	Trustees Of Boston University	Conversion d'adn avec conservation de séquence
WO2012042052A1 (fr) *	2010-09-30	2012-04-05	Geneseque As	Procédé de séquençage

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
BRENNER SYDNEY ET AL: "Gene expression analysis by massively parallel signature sequencing (MPSS) on microbead arrays", NATURE BIOTECHNOLOGY, NATURE PUBLISHING GROUP, NEW YORK, NY, US, vol. 18, no. 6, 1 June 2000 (2000-06-01), pages 630 - 634, XP002199492, ISSN: 1087-0156, DOI: 10.1038/76469 *
HEIKO KUHN ET AL: "Template-independent ligation of single-stranded DNA by T4 DNA ligase", FEBS JOURNAL, vol. 272, no. 23, 16 December 2005 (2005-12-16), pages 5991 - 6000, XP055084765, ISSN: 1742-464X, DOI: 10.1111/j.1742-4658.2005.04954.x *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number	Priority date	Publication date	Assignee	Title
WO2016020292A1 (fr) *	2014-08-06	2016-02-11	Geneseque As	Procédé de séquençage de polynucléotides immobilisés
US11549141B2 (en)	2014-08-06	2023-01-10	Geneseque As	Method of sequencing immobilized polynucleotides

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US10876158B2 (en)	2020-12-29	Method for sequencing a polynucleotide template
US20230098456A1 (en)	2023-03-30	Methods for sequencing a polynucleotide template
US20220042090A1 (en)	2022-02-10	PROGRAMMABLE RNA-TEMPLATED SEQUENCING BY LIGATION (rSBL)
CN114901818B (zh)	2025-01-10	靶向核酸文库形成的方法
JP5653964B2 (ja)	2015-01-14	錠型（ｐａｄｌｏｃｋ）プローブのローリングサークル複製
EP2691546B1 (fr)	2017-07-12	Identification d'une matrice d'acides nucléiques dans une réaction de séquençage multiplex
AU2001271816B9 (en)	2007-05-24	Polynucleotide sequence assay
EP1910560B1 (fr)	2010-12-29	Procede de sequencage d'une matrice de polynucleotide
US6258539B1 (en)	2001-07-10	Restriction enzyme mediated adapter
US20220364169A1 (en)	2022-11-17	Sequencing method for genomic rearrangement detection
CA3213538A1 (fr)	2015-12-10	Methode d'identification et d'enumeration de changements en matiere de sequence d'acide nucleique, expression, copie ou methylation d'adn en utilisant des reactions associant nucl ease, ligase, polymerase et sequencage
US20090191563A1 (en)	2009-07-30	Uniform fragmentation of dna using binding proteins
EA012525B1 (ru)	2009-10-30	Способ подготовки полинуклеотидов для анализа
WO2009032167A1 (fr)	2009-03-12	Procédé de séquençage d'une matrice de polynucléotides
JPH10508483A (ja)	1998-08-25	オリゴヌクレオチドプローブまたはプライマーの製法、そのためのベクターおよびそれらの使用
CN110446791B (zh)	2024-04-16	多核苷酸衔接子及其使用方法
DK2456892T3 (en)	2014-12-08	Procedure for sequencing of a polynukleotidskabelon
WO2014033285A1 (fr)	2014-03-06	Méthode de séquençage pour polynucléotide monobrin utilisant une sonde avec site de reconnaissance pour nucléase
WO2025062002A1 (fr)	2025-03-27	Séquençage simultané à l'aide d'une traduction de coupure simple brin

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