+

WO2008032058A2 - Procédé de séquençage d'un polynucléotide - Google Patents

Procédé de séquençage d'un polynucléotide Download PDF

Info

Publication number
WO2008032058A2
WO2008032058A2 PCT/GB2007/003451 GB2007003451W WO2008032058A2 WO 2008032058 A2 WO2008032058 A2 WO 2008032058A2 GB 2007003451 W GB2007003451 W GB 2007003451W WO 2008032058 A2 WO2008032058 A2 WO 2008032058A2
Authority
WO
WIPO (PCT)
Prior art keywords
polynucleotide
target
concatemer
hybridised
sequence
Prior art date
Application number
PCT/GB2007/003451
Other languages
English (en)
Other versions
WO2008032058A3 (fr
Inventor
Preben Lexow
Original Assignee
Lingvitae As
Jappy, John, William, Graham
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Lingvitae As, Jappy, John, William, Graham filed Critical Lingvitae As
Publication of WO2008032058A2 publication Critical patent/WO2008032058A2/fr
Publication of WO2008032058A3 publication Critical patent/WO2008032058A3/fr

Links

Classifications

    • 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

  • This invention relates to methods for determining the sequence of polynucleotides.
  • WO-A-00/39333 describes a method for sequencing polynucleotides by converting the sequence of a target polynucleotide into a second polynucleotide having a defined sequence and positional information contained therein.
  • the sequence information of the target is said to be "magnified” in the second polynucleotide, allowing greater ease of distinguishing between the individual bases on the target molecule.
  • This is achieved using "magnifying tags", which are predetermined units of nucleic acid sequence.
  • Each of the bases adenine, cytosine, guanine and thymine on the target molecule is represented by an individual magnifying tag, converting the original target sequence into a magnified sequence. Conventional techniques may then be used to determine the order of the magnifying tags, and thereby determine the specific sequence on the target polynucleotide.
  • each magnifying tag comprises a label, e.g. a fluorescent label, which may then be identified and used to characterise the magnifying tag.
  • a label e.g. a fluorescent label
  • each magnifying tag comprises two units of distinct sequence which can be used as a binary system, with one unit representing "0" and the other representing "1".
  • Each base on the target is characterised by a combination of the two units, for example adenine may be represented by "0" + “0”, cytosine by "0” +"1", guanine by "1” + “0” and thymine by "1" +"1".
  • the present invention provides a method for analysing polynucleotides.
  • the method utilises a concatemer of the target polynucleotide, i.e. repeating the sequence of the target polynucleotide, and then interrogating the various target polynucleotides to reveal the target polynucleotide sequence.
  • the intention is, preferably, to identify one base (nucleotide) of each target polynucleotide on the concatemer with different bases being identified for each target. In this way, all the bases to be identified are more separated than if the bases of the original target polynucleotide were to be sequenced. Increasing the separation allows the eventual read-out technology to discriminate between the units, thereby improving the efficiency of the eventual sequencing/identification step.
  • a method for sequencing a target polynucleotide comprises the steps of:
  • step (ii) forming a concatemer comprising multiple copies of the product of step (i);
  • a method for sequencing a target polynucleotide comprises the steps of:
  • a support surface comprises a double-stranded polynucleotide immobilised thereon, wherein one strand is a concatemer of repeating polynucleotide sequences having regions hybridised to the other strand and non-hybridised regions.
  • Figure 1 illustrates the use of a circular polynucleotide to generate the concatemer of the target polynucleotide
  • Figure 2 illustrates the hybridisation of a (third) polynucleotide to the sequence adjacent to the non-hybridised target, permitting interrogation with a labelled ddNTP, and
  • Figure 3 shows the subsequent incorporation of a labelled ddNTP.
  • polynucleotide is well known in the art and is used to refer to a series of linked nucleic acid molecules, e.g. DNA or RNA.
  • Nucleic acid mimics e.g. PNA, LNA (locked nucleic acid) and 2 -O-methRNA are also within the scope of the invention.
  • bases A, T(U), G and C relate to the nucleotide bases adenine, thymine (uracil), guanine and cytosine, as will be appreciated in the art.
  • Uracil replaces thymine when the polynucleotide is RNA, or it can be introduced into DNA using dUTP, again as well understood in the art.
  • first polynucleotide is used herein to refer to a polynucleotide of known sequence and length which is used to ligate to the target, preferably to circularise the ligated target.
  • the first polynucleotide acts to provide separation between different copies of the target on the eventual concatemer.
  • the target polynucleotide is linked at either its 5 1 or 3' end to the first polynucleotide, preferably at both the 5 1 and 3 1 ends to form the circular product.
  • second polynucleotide is used herein to refer to a polynucleotide intended to hybridise to regions of a concatemer formed with repeated target and first polynucleotide sequences.
  • the second polynucleotide may also be referred to as a "masking" polynucleotide as it acts to prevent interrogation of those regions of the first polynucleotide to which it hybridises.
  • the regions of the first polynucleotide that are not hybridised are said to be "unmasked".
  • the second polynucleotide therefore comprises a repeated sequence complementary to the first polynucleotide, interspersed with a sequence which does not hybridise to either the target or the first polynucleotide. This ensures that the target sequence (or at least a portion of the target sequence) does not hybridise to the second polynucleotide and is therefore available for interrogation in a subsequent step.
  • the second polynucleotide also has a sequence that does not hybridise to a portion of the sequence of the first polynucleotide adjacent to the target.
  • This will be of known sequence and permits the hybridisation of a third polynucleotide sequence adjacent to the target using the second polynucleotide as its complement.
  • This portion of the second polynucleotide will usually be downstream of the target and will typically be from 10 to 40 bases in size, more typically from 15 to 25 bases, and most typically 20 bases. This provides sufficient discrimination for the hybridisation to the third polynucleotide sequence.
  • the method of the present invention is used to convert a single target polynucleotide sequence into a series of polynucleotides which can each be interrogated at intervals more spaced apart than that of a single target. This has the benefit of, in effect, separating the bases on the target to permit the ultimate interrogation and read-out steps to be performed with more accuracy and discrimination.
  • the invention relies on the formation of a concatemer of the target polynucleotide which permits subsequent interrogation to be performed on selected bases; the interrogated bases are representative of the bases on the original target polynucleotide.
  • one or more of the bases of the target polynucleotide can be interrogated in various ways to reveal their identity.
  • the intention may be to determine the full or partial sequence of the target. Separate individual bases on each target of the concatemer may be targeted and identified. Alternatively, the intention may be to determine a single base on the target, with the multiple targets of the concatemer being used as controls, to ensure that the identified signal is correct. This may be of use in determining single nucleotide polymorphisms (SNPs).
  • SNPs single nucleotide polymorphisms
  • all bases on the original target are identified ultimately, with a single base being identified on each interrogated target on the concatemer. It is preferable if a single base is interrogated (i.e. determined) within a single target on the concatemer, and different bases are interrogated on different copies of the target.
  • the preferred way of interrogating the concatemer is to hybridise one or more third polynucleotides of defined sequence to the concatemer such that the third polynucleotide hybridises to the region adjacent to the target, permitting interrogation of the base next to it, to occur.
  • Different target sequences on the concatemer can be interrogated at different base positions by modifying the size of the third polynucleotide, as shown in Figures 2 and 3.
  • Universal bases can be added to the third polynucleotides so that the different lengths can be achieved at the same time as retaining the hybrid. This permits control over which base on the target is to be interrogated.
  • the different sized third polynucleotides can be added sequentially or together. The concentration of each third polynucleotide can be controlled to ensure that each binds to a target. If added together, the different sized third polynucleotides can be labelled so that a distinction can be made for each reaction.
  • the second polynucleotide (masking polynucleotide) can be designed so that there is a different sequence masking each target region such that this different sequence can be used to hybridise different third polynucleotides depending on the position to be interrogated. It will therefore be possible design different primers depending on which position is to be targeted. These can be added sequentially to carry out interrogation.
  • the base(s) can be interrogated by carrying out a polynucleotide extension reaction, using dideoxy nucleotides (ddNTP) that are detectably labelled.
  • ddNTP dideoxy nucleotides
  • the third polynucleotide, acting as a primer, is thereby extended by one base, which can be detected. Further extension is prevented due to the use of the ddNTP which does not permit further extension.
  • the ddNTPs can be labelled in any convenient way, but preferably are labelled with a fluorophore; a different type for each of the different ddNTPs.
  • the labelling of nculeotides with fluorophores is now widely known in the art, and conventional reagents and procedures can be used.
  • the target sequence is ligated to the known sequence of the first polynucleotide prior to concatemerisation, so that the concatemer comprises both target polynucleotide sequences and known sequences, so that hybridisation can occur between the concatemer and the second polynucleotide.
  • the known sequences of the first polynucleotide should be of sufficient length to permit hybridisation with the second polynucleotide to occur.
  • the known sequences should be more than 100 nucleotides, preferably more than 500 nucleotides. This provides separation between the hybridised sequences and the non- hybridised (target) sequences, which can then be interrogated.
  • the target and first polynucleotide are circularised to aid the formation of the concatemer.
  • the target may be circularised in any convenient way.
  • the single-stranded target is hybridized to the 3' end of the first polynucleotide. Both the 5' and the 3' end of the target molecule will hybridize to the first polynucleotide and will be ligated together forming a single-stranded circle.
  • the efficiency of circle ligations is much better with increased complementarity and it is preferred to use at least 6 complementary nucleotides, preferably at least 9 complementary nucleotides for hybridisation to the first polynucleotide.
  • the ligase can be any available ligase, but is preferably T4 DNA ligase, E.coli DNA ligase or Taq DNA ligase.
  • a support-bound oligonucleotide can be used to hybridise to the target and to ligate to the first polynucleotide.
  • the hybrid forms a partially double-stranded molecule with an overhang complementary to the first polynucleotide's 3' end.
  • the support oligonucleotide can then be ligated to the first polynucleotide at the 3' end.
  • the 5' end of the target is also complementary to the first polynucleotide and so the target will hybridise to the first polynucleotide bringing the two ends of the target into position for a ligase to join the two ends of the target, forming a circle.
  • the support oligonucleotide acts to help retain the now circularised target at the first polynucleotide, ready for concatemerisation.
  • the support-bound oligonucleotide will be of a size sufficient to aid hybridisation and circularisation with the target.
  • the target polynucleotide can be concatemerised in any convenient way.
  • a polymerase reaction is used.
  • the circularised target (first) polynucleotide acts as a template for a polymerase reaction.
  • the template is a circular molecule, the technique used is commonly known as Rolling Circle Amplification (RCA).
  • RCA Rolling Circle Amplification
  • Linear RCA utilises one primer, producing one concatemer from each template.
  • Exponential RCA utilises two primers, where one is complementary to the target to be amplified, while the other is complementary to the product generated by the first primer.
  • the second primer initiates the synthesis of multiple concatemerised copies from one target polynucleotide.
  • Multiply- primed RCA utilises a set of random hexamers as primers. These primers initiate the synthesis of multiple concatemerised copies from one target polynucleotide. Secondary non-specific priming events can occur subsequently on the displaced product strands of the initial RCA step.
  • polymerases can be used, including Sequenase, Bst DNA polymerase (large fragment), Klenow exo-DNA polymerase, which are all polymerases operating at 37oC and displaying the strand displacement ability which is preferable for making the concatemers.
  • the heat-stable Vent exo-DNA polymerase may be used.
  • the enzyme shown in the literature to be most efficient on acting on circular templates is phi29 polymerase, and this is preferred. Concatemerisation may also be carried out in ways not dependant on
  • RCA complementary metal-oxide-semiconductor
  • multiple copies of the target/first polynucleotide can be ligated together using conventional methods, to form a concatemerised product.
  • Other ways will also be evident to the skilled person.
  • Hybridisation with mask (second) polynucleotide The hybridisation to the second polynucleotide can be carried out directly as the concatemer is produced. Accordingly, the second polynucleotide can be present during the formation of the concatemer.
  • the circular target may be attached (ligated) to the second polynucleotide, so that the polymerase product is formed in proximity to the second polynucleotide, aiding hybridisation.
  • the hybridisation can also be separated from the concatemerisation reaction by "blocking" the second polynucleotide using a complementary molecule.
  • the blocking molecule can either be synthesised in a separate reaction and then annealed to the second polynucleotide prior to the concatemerisation reaction.
  • the blocking molecule can be synthesised by a polymerase directly on the second polynucleotide by using a short primer.
  • the blocking molecule can be removed using an exonuclease, and the second polynucleotide is then available for hybridisation to the concatemerised target molecule and its polymerised product.
  • the second polynucleotide will have at least partial complementarity to the concatemer. This is achieved by knowledge of the first polynucleotide. The intention is to hybridise the sequence corresponding to the first polynucleotide to the second polynucleotide, such that there are non- hybridised portions corresponding to the target sequence which can be interrogated. Interrogation of the resulting hybrid may be carried out using any convenient read-out technique.
  • the present invention also relates to support materials which comprise the polynucleotides defined herein.
  • a support surface will comprise a double- stranded polynucleotide immobilised thereon, wherein one strand will be the concatemer molecule and the other strand will be the second polynucleotide, wherein there are hybridised and non-hybridised regions.
  • Any suitable support material may be used, including conventional glass, ceramic or plastics materials having a suitable surface.
  • Immobilisation may be carried out using conventional techniques and covalent and non-covalent attachment may be used.
  • it is the second polynucleotide that is covalently attached to the support.
  • Suitable linker molecules will be apparent to the skilled person. The content of all the publications referred to herein are incorporated herein by reference.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Immunology (AREA)
  • Biotechnology (AREA)
  • Molecular Biology (AREA)
  • Biophysics (AREA)
  • Analytical Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

La présente invention concerne un procédé destiné au séquençage d'un polynucléotide cible, consistant: (i) à ligaturer le polynucléotide cible à un premier polynucléotide; (ii) à former un concatémère comprenant plusieurs copies du produit issu de l'étape (i); (iii) à fixer le concatémère à un second polynucléotide de sorte qu'il y ait hybridation entre le second polynucléotide et des parties du concatémère, mais pas des régions sur le concatémère correspondant à au moins une partie du polynucléotide cible; et (iv) à interroger une ou plusieurs bases dans ces régions non hybridées avec le second polynucléotide, afin d'identifier la séquence polynucléotidique cible.
PCT/GB2007/003451 2006-09-13 2007-09-13 Procédé de séquençage d'un polynucléotide WO2008032058A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB0618046.7 2006-09-13
GB0618046A GB0618046D0 (en) 2006-09-13 2006-09-13 Method

Publications (2)

Publication Number Publication Date
WO2008032058A2 true WO2008032058A2 (fr) 2008-03-20
WO2008032058A3 WO2008032058A3 (fr) 2008-10-09

Family

ID=37309875

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/GB2007/003451 WO2008032058A2 (fr) 2006-09-13 2007-09-13 Procédé de séquençage d'un polynucléotide

Country Status (2)

Country Link
GB (1) GB0618046D0 (fr)
WO (1) WO2008032058A2 (fr)

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014145296A3 (fr) * 2013-03-15 2014-11-27 Theranos, Inc. Amplification d'acide nucléique
WO2015076919A1 (fr) * 2013-11-22 2015-05-28 Theranos, Inc. Amplification des acides nucléiques
US9416387B2 (en) 2013-03-15 2016-08-16 Theranos, Inc. Nucleic acid amplification
US9916428B2 (en) 2013-09-06 2018-03-13 Theranos Ip Company, Llc Systems and methods for detecting infectious diseases
US10450595B2 (en) 2013-03-15 2019-10-22 Theranos Ip Company, Llc Nucleic acid amplification
US11254960B2 (en) 2013-03-15 2022-02-22 Labrador Diagnostics Llc Nucleic acid amplification

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001020039A2 (fr) * 1999-09-16 2001-03-22 Curagen Corporation Methode de sequençage d"un acide nucleique
US20030215821A1 (en) * 1999-04-20 2003-11-20 Kevin Gunderson Detection of nucleic acid reactions on bead arrays
US20040110153A1 (en) * 2002-12-10 2004-06-10 Affymetrix, Inc. Compleixity management of genomic DNA by semi-specific amplification

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030215821A1 (en) * 1999-04-20 2003-11-20 Kevin Gunderson Detection of nucleic acid reactions on bead arrays
WO2001020039A2 (fr) * 1999-09-16 2001-03-22 Curagen Corporation Methode de sequençage d"un acide nucleique
US20040110153A1 (en) * 2002-12-10 2004-06-10 Affymetrix, Inc. Compleixity management of genomic DNA by semi-specific amplification

Cited By (19)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10017809B2 (en) 2013-03-15 2018-07-10 Theranos Ip Company, Llc Nucleic acid amplification
US10745745B2 (en) 2013-03-15 2020-08-18 Labrador Diagnostics Llc Nucleic acid amplification
US9416387B2 (en) 2013-03-15 2016-08-16 Theranos, Inc. Nucleic acid amplification
US10131939B2 (en) 2013-03-15 2018-11-20 Theranos Ip Company, Llc Nucleic acid amplification
US9551027B2 (en) 2013-03-15 2017-01-24 Theranos, Inc. Nucleic acid amplification
CN106715710A (zh) * 2013-03-15 2017-05-24 赛拉诺斯股份有限公司 核酸扩增
US9725760B2 (en) 2013-03-15 2017-08-08 Theranos, Inc. Nucleic acid amplification
US11649487B2 (en) 2013-03-15 2023-05-16 Labrador Diagnostics Llc Nucleic acid amplification
US11603558B2 (en) 2013-03-15 2023-03-14 Labrador Diagnostics Llc Nucleic acid amplification
CN106715710B (zh) * 2013-03-15 2022-10-25 赛拉诺斯知识产权有限责任公司 核酸扩增
US11254960B2 (en) 2013-03-15 2022-02-22 Labrador Diagnostics Llc Nucleic acid amplification
US10450595B2 (en) 2013-03-15 2019-10-22 Theranos Ip Company, Llc Nucleic acid amplification
WO2014145296A3 (fr) * 2013-03-15 2014-11-27 Theranos, Inc. Amplification d'acide nucléique
US10522245B2 (en) 2013-09-06 2019-12-31 Theranos Ip Company, Llc Systems and methods for detecting infectious diseases
US10283217B2 (en) 2013-09-06 2019-05-07 Theranos Ip Company, Llc Systems and methods for detecting infectious diseases
US9916428B2 (en) 2013-09-06 2018-03-13 Theranos Ip Company, Llc Systems and methods for detecting infectious diseases
CN106255763A (zh) * 2013-11-22 2016-12-21 赛拉诺斯股份有限公司 核酸扩增
CN106255763B (zh) * 2013-11-22 2022-06-24 赛拉诺斯知识产权有限责任公司 核酸扩增
WO2015076919A1 (fr) * 2013-11-22 2015-05-28 Theranos, Inc. Amplification des acides nucléiques

Also Published As

Publication number Publication date
GB0618046D0 (en) 2006-10-25
WO2008032058A3 (fr) 2008-10-09

Similar Documents

Publication Publication Date Title
US20090053699A1 (en) Method for Preparing Polynucleotides for Analysis
EP0868530B1 (fr) Reaction d'amplification en cascade d'acide nucleique
US5955276A (en) Compound microsatellite primers for the detection of genetic polymorphisms
EP0777747B1 (fr) Procede de sequen age de nucleotides
EP1021562B1 (fr) Amplification par deplacements multiples
US7414115B2 (en) Length determination of nucleic acid repeat sequences by discontinuous primer extension
US20070269825A1 (en) Method and kit for nucleic acid sequence detection
EP1613773B1 (fr) Procede de caracterisation de polynucleotides
DE50113454D1 (de) Verfahren zur bestimmung des methylierungsgrades von bestimmten cytosinen in genomischer dna im sequenzkontext 5'-cpg-3'
JP2005515792A (ja) 核酸を操作するための方法および手段
EP1173612B1 (fr) METHODE DE DETECTION ET/OU D'ANALYSE, AU MOYEN DE TECHNIQUES D'EXTENSION D'AMORCES, DE POLYMORPHISMES NUCLEOTIDIQUES UNIQUES DANS DES FRAGMENTS DE RESTRICTION, ET NOTAMMENT DANS DES FRAGMENTS DE RESTRICTION AMPLIFIES GENERES PAR AFLP$m(3)
WO2008032058A2 (fr) Procédé de séquençage d'un polynucléotide
JP2006508677A (ja) 遺伝子発現のオリゴヌクレオチド誘導分析
WO2002024960A1 (fr) Detection de la variation d'adn

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 07804245

Country of ref document: EP

Kind code of ref document: A2

NENP Non-entry into the national phase in:

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 07804245

Country of ref document: EP

Kind code of ref document: A2

点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载