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WO2014170084A1 - Procédé d'extraction d'au moins un fragment d'acide nucléique bicaténaire - Google Patents

Procédé d'extraction d'au moins un fragment d'acide nucléique bicaténaire Download PDF

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Publication number
WO2014170084A1
WO2014170084A1 PCT/EP2014/055562 EP2014055562W WO2014170084A1 WO 2014170084 A1 WO2014170084 A1 WO 2014170084A1 EP 2014055562 W EP2014055562 W EP 2014055562W WO 2014170084 A1 WO2014170084 A1 WO 2014170084A1
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WO
WIPO (PCT)
Prior art keywords
nucleic acid
nanochannel
double
cutting
acid fragment
Prior art date
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/EP2014/055562
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German (de)
English (en)
Inventor
Oliver Hayden
Saskia Rausch
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Siemens AG
Siemens Corp
Original Assignee
Siemens AG
Siemens Corp
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 Siemens AG, Siemens Corp filed Critical Siemens AG
Publication of WO2014170084A1 publication Critical patent/WO2014170084A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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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
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y15/00—Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors

Definitions

  • the invention relates to a method for obtaining at least one double-stranded nucleic acid fragment.
  • next Generation sequencing methods allow the Se quenzierung of millions of DNA fragments in a single process pass through massive parallelization of the individual steps.
  • the Sequen ⁇ z istsvon double stranded nucleic acid fragments having a uniform length defined require.
  • genomic DNA the DNA is first cut to ⁇ due, for example by means of ultrasound, resulting in DNA fragments of different lengths, from which the fragments with matching uniform length must be subsequently isolated in additional steps for example the
  • the invention is therefore based on the object to provide a method by which a double-stranded nucleic acid can be cut into identically long nucleic acid fragments.
  • the object is achieved by a method having the features according to claim 1.
  • the method according to the invention for obtaining at least one double-stranded nucleic acid fragment comprises the steps of: providing at least one nanochannel loaded with a single double-stranded nucleic acid, electrically cutting the nucleic acid to generate a double strand break, to form at least one double-stranded nucleic acid to obtain an acidic fragment, and recovering the nucleic acid fragment from the nanochannel.
  • the nucleic acid may be an RNA or a DNA. Naturally occurring nucleic acids and chemically produced or recombinantly produced nucleic acids are preferred.
  • the nucleic acid preferably has a length of about 1,000 base pairs to about 100,000,000 base pairs.
  • the nucleic acid fragment preferably has a length of about 10 base pairs to about 10,000 base pairs, more preferably from about 50 base pairs to about 500 base pairs.
  • nanochannel refers to a channel having a horizontal extension of a few nanometers to a few micrometers.
  • the horizontal extent of the nanochannel is for example between about 5 nm and about 150 nm of the nano channel includes before ⁇ preferably a vertical extension of less than 300. nm. the nanochannel for example, has a vertical He ⁇ elongation of 50 nm. the length of the nano channel may vary depending on the application. as the material, preferably silicon or glass-like substrates such as quartz glass are used for the nanochannel.
  • the nanochannel is loaded with a single double-stranded nucleic acid.
  • double-stranded nucleic acids are in solution in a compact, random wound ( "coiled") form, similar to a ball.
  • the compact secondary structure of the nucleic acid spontaneously unfolds and extends.
  • This physical aspect of the nuc ⁇ leinklare means that the nucleic acid in the nanochannel assumes a stretched linear form. By the linear form are all portions of the nucleic acid equal a machining ⁇ tung accessible.
  • the extension prevents the nucleic acid in the nanochannel from folding back on itself or intertwining.
  • the loading of the nanochannel with the nucleic acid takes place at ⁇ example by applying an electric field having at ⁇ play, 1 to 50 V / cm, by capillary forces, by differential surface tension, by a chemical see gradient or by a differential pressure conditions ⁇ nis, which is generated for example by applying a vacuum.
  • an electrical cutting of the nucleic acid for the generation ⁇ supply of a double-strand break is made to at least one
  • Loading ⁇ handle "electrical cutting” refers to cutting the nucleic acid by means of electricity, wherein the covalent che ⁇ mix bond between two adjacent nucleotides in each of the two nucleic acid strands of the double strand is broken down locally. Therefore, the cutting produces a double-strand break in the nucleic acid . This is obtained at least a double-stranded nucleic acid fragment.
  • the nucleic acid fragment ⁇ is therefore a drawschnit ⁇ tenes of the nucleic acid portion that is obtained in a further step of the method of the nanochannel.
  • the electrical cutting of the nucleic acid is preferably carried out by exposing the nucleic acid to an electric field, which is generated for example by applying an electrical voltage to the region of the nanochannel at which the cutting of the nucleic acid is to take place.
  • the electric field exerts locally from a sufficiently high force to the nucleic acid such that the double strand of the nucleic acid ⁇ breaks.
  • the force to the nucleic acid to separate a phosphodiester in the nucleic acid to by ⁇ , greater than 1 nano Newton is preferred.
  • this has electric field has an electric field strength of about 25 kV / m to about 250 kV / m.
  • the nucleic acid fragment is recovered from the nanochannel. This is preferably done in a simple manner, such as, for example, by purging the nucleic acid fragment from the nanochannel.
  • the nucleic acid fragment can be obtained from the Na ⁇ nokanal, for example, by applying an electric field or a chemical gradient.
  • the recovered nucleic acid fragment has a certain defi ned ⁇ length. The length of the nucleic acid fragment can be controlled by selecting suitable conditions for cutting the nucleic acid.
  • An advantage of the method of the invention is that a nucleic acid fragment having a determinable length Herge ⁇ represents is. This further makes it possible to cut a nucleic acid into several nucleic acid fragments of the same length. Such nucleic acid fragments can be used beispielswei ⁇ se for sequencing. Additional Ar ⁇ beits Coloure for isolating nucleic acid fragments of appropriate length from a mixture of different length nucleic acid fragments are not required. This causes a loss of nucleic acid material, which is connected to the additional Ar ⁇ beits suitsen avoided. Furthermore, time and costs are saved.
  • Nucleotide sequence of the nucleic acid takes place. Thus, independent of the nucleic acid sequence, multiple nucleic acid fragments of uniform length can be obtained. This makes it possible to use the method also for cutting a nucleic acid of unknown sequence. Especially with nucleic acids to be sequenced, the sequence is typically partially or completely unknown.
  • the steps of the method according to the invention are preferably carried out in the order as given in claim 1. The steps can also be carried out simultaneously. In a preferred embodiment, the
  • Steps b) and c) of the method according to the invention combined.
  • the method can be performed particularly zeitspa ⁇ rend.
  • the nanochannel has a width of 10 nm and a depth of 50 nm. This Dimensio ⁇ nen lead to a steady unfolding of the nucleic acid in the nanochannel.
  • the nucleic acid is a DNA, preferably a genomic DNA.
  • DNA is used for a variety of molecular biological applications.
  • genomic DNA is often the starting material for sequencing procedures.
  • so-called "next generation" sequencing methods which can sequence millions of DNA fragments in a single sequencing run, require DNA fragments of a uniform length. The method according to the invention makes it possible to provide such DNA fragments in a particularly simple and rapid manner .
  • the nucleic acid is an RNA. Double-stranded RNA is found among other things in certain viruses.
  • the method according to the invention can be used to study at least one of the viral biology or diseases caused by the virus
  • the inventive method can also be used with a
  • single-stranded nucleic acid to obtain at least one single-stranded nucleic acid fragment.
  • the nanochannel is loaded at a first end with the nucleic acid and the nucleic acid Acid fragment is recovered at a second end of the nanochannel. This does not escape from the extraction of nucleic acid fragment from the nanochannel also the Nuk ⁇ leinklare from the nanochannel. A mixture of the nucleic acid fragment with the nucleic acid is prevented. For the masters ⁇ th further uses of the nucleic acid fragments must be separated from the nucleic acid. Furthermore, the loading of the nanochannel prevented at the first end with the nucleic ⁇ ynoic acid and recovering the nucleic acid fragment at the two-th end of the nanochannel that the nucleic acid fragment from the extraction of the nanochannel by the nucleic acid is sterically hindered. In addition, the inventive method is particularly easy in this way and efficiently carried out ⁇ the. As a result, the method is suitable for high
  • the cutting of the nucleic acid takes place in the nanochannel.
  • the conditions for the electrical cutting of the nucleic acid can be set particularly precisely because they are independent of external influences.
  • the cutting of the nucleic acid takes place at the second end of the nanochannel.
  • the nucleic acid is thus cut only at the point where it exits the nanochannel.
  • the nucleic acid is cut in a region of the nanochannel in which the nucleic acid is in an elongated form.
  • the term "elgonator form” refers to a stretched, linear structural ⁇ structure of the nucleic acid.
  • the nucleic acid is unfolded and takes an elongated Shape.
  • the range of the nanochannel, in which the nucleic acid is present in the form of elongated not already un ⁇ indirectly the nanochannel at the first end. Rather, the range begins at a certain distance from the first end of the nanochannel.
  • the length of the distance depends inter alia on the dimensions of the nanochannel and the conditions when loading the nanochannel with the nucleic acid.
  • the cutting of the nucleic acid in a region of the nanochannel, in the present nuc ⁇ leinklare in elongated shape ensures that the double-strand break is generated only at a specific site of the nucleic acid without additional single-stranded ⁇ breaks occur at the nucleic acid.
  • Single strand breaks occur at Kings ⁇ NEN nucleic acid portions overlying the site to be cut of the nucleic acid, when the cutting is performed in a region of the nanochannel, in which the nucleic acid is not present completely unfolded. This to ⁇ slegilichen single-strand breaks can interfere in the further use of the nucleic acid fragment.
  • At least two nucleic acid fragments are obtained, which have an equal length on ⁇ .
  • ⁇ handle "length" refers to the number of base pairs of nucleic ynoic acid fragments or alternatively, the metric length of the
  • Nucleic acid fragments for example, in nm. Nucleic acid fragments of equal length may be used for a variety of molecular biology applications requiring uniformly long nucleic acid fragments. Examples of play, the nucleic acid fragments are used for a Se ⁇ quenzierung by means of a "Next Generation" ⁇ sequencing methods. In this case, additional work steps are avoided ⁇ fragments for isolating nucleic acid fragments of appropriate length from a mixture of different lengths nucleic acid.
  • the nucleic acid and / or the nucleic acid fragment are translocated through the nanochannel. ported.
  • Preferably transporting the nucleic ⁇ ynoic acid and / or of the nucleic acid fragment along the length of the nanochannel is carried out, particularly preferably from the first end of the nanochannel directed to the second end of the nanochannel.
  • the method can be carried out particularly efficiently. This is particularly true for the case that several nucleic acid fragments to be generated from one and dersel ⁇ ben nucleic acid.
  • the nucleic acid fragments on the second end of the nanochannel are transported from the nanochannel out at the same time and thereby ge ⁇ gained, while the nucleic acid is prepared starting from the first end of the nanochannel transported through the nanochannel in the direction of the second end.
  • the nucleic acid may be cut once again so that another nucleic acid fragment of the same nucleic acid ⁇ formed.
  • the nucleic acid and the nucleic acid fragment are transported through the nanochannel at the same rate. This prevents that the nucleic acid be ⁇ prevents the transport of the nucleic acid fragment by the nanochannel sterically or vice versa.
  • Transporting the nucleic acid and / or the nucleic acid fragment can be carried out, for example, by rinsing the nucleic ⁇ re and / or the nucleic acid fragment by the nanochannel by applying a liquid stream.
  • the transport may also be accomplished by applying a capillary flow, a chemical gradient, a magnetic field, a mechanical force, or a pressure gradient, or by a combination thereof.
  • the transport of the nucleic acid and / or the nucleic acid fragment is carried out electrophoretically by applying an electric field.
  • the electric field may, for example, 1 to 50 V / cm have ⁇ .
  • the nucleic acid and / or the nucleic acid acidic fragment are transported at a desired speed in ei ⁇ ne desired direction through the nanochannel.
  • the nanochannel is combined with a microscopic device through which the cutting of the nucleic acid and / or transport of the nucleic ⁇ ynoic acid and / or of the nucleic acid fragment can be monitored.
  • the transport of the nucleic acid and / or the nucleic acid fragment through the nanochannel occurs at a constant rate. This makes it particularly easy to control and / or monitor the cutting of the nucleic acid and / or the recovery of the nucleic acid fragment.
  • the cutting of the nucleic acid is timed. After each cut of the nucleic acid passes a defined period of time before further cutting of the nucleic acid takes place.
  • the period may be, for example, about 1 second to about 1 minute.
  • the temporal clocking is selected as a function of the desired length of the nucleic acid fragment and can furthermore be selected as a function of the speed with which the nucleic acid is transported through the nanochannel.
  • the nucleic acid is transported at a constant speed through the nanochannel, during a time clocked cutting the nucleic acid in a region of the nanochannel, in which the nuc ⁇ leinklare present in an elongated shape is carried out.
  • the nucleic acid is transported at a constant speed through the nanochannel, during a time clocked cutting the nucleic acid in a region of the nanochannel, in which the nuc ⁇ leinklandre present in an elongated shape is carried out.
  • the electrical cutting of the nucleic acid takes place preference ⁇ example in that the nucleic acid is subjected to an electric field. In a preferred embodiment, the electric field is applied in the form of a pulse. Thereby the cutting of the nucleic acid can be gesteu ⁇ ert particularly precise.
  • the pulse preferably has a duration in the range of microseconds to milliseconds and / or a frequency of 0.5 Hz to about 100 MHz.
  • the pulsed electric field preferably has an electric field strength of more than 100 kV / m.
  • the pulse may have a frequency of 1 Hz, a duration of 100 ys, and an electric field strength of 250 kV / m. Due to the short pulse duration in the range of ys, a strong, for the device, the nucleic acid and / or the nucleic acid fragment harmful heat generation can be avoided even at high electric field strengths.
  • the pulse has a frequency of 1 Hz, a duration of 80 ms and an electric field strength of 50 kV / m.
  • the longer pulse duration in the range of ms has the advantage that at a constant frequency, a ge ⁇ rings electric field strength sufficient to produce the double-strand break ⁇ in the nucleic acid.
  • the electrical cutting of the nucleic acid takes place in at least two spatially discrete regions of the nanochannel, preferably in three to about ten spatially distinct regions of the nanochannel.
  • the nucleic acid can be cut simultaneously several times, so that several nucleic acid fragments are obtained simultaneously.
  • the throughput of the method can be increased.
  • a particularly time-saving and cost-effective implementation of the method, even at high throughput allows.
  • the nanochannel is automatically loaded with nucleic acid and concentrated ⁇ sets over multiple process cycles ⁇ for a high-throughput variant of the method.
  • at least two nanochannels are provided.
  • nanochannels are arranged, for example, parallel to one another and / or stacked one above the other.
  • the process is carried out in parallel in the nanochannels so that the respective nucleic acids in the nanochannels are cut simultaneously.
  • the throughput of the method can be increased.
  • the time required to recover nucleic acid fragments from at least two nucleic acids is significantly reduced as compared to using only one nanochannel.
  • the nanochannel is part of a nanochannel array or of a nanofluidic or microfluidic system.
  • FIG. 1 shows a gain of three double-stranded nucleic acid fragments 9 by means of the method according to the invention.
  • a nanochannel 1 with a first end 3 and egg ⁇ nem second end 5 is provided, which is loaded with a single double-stranded nucleic acid 7.
  • the nanochannel 1 has a horizontal extent of 10 nm and a verti ⁇ cal extent of 50 nm.
  • the surface of the nanochannel 1 is made of silicon dioxide.
  • the nucleic acid is a 7 From ⁇ cut genomic DNA.
  • the DNA is flushed to the second end 5 of the nanochannel 1 through the nanochannel 1 at a constant rate from the first end 3 of the nanochannel 1, where the nanochannel 1 was loaded with the DNA.
  • the DNA corresponds folds in the nanochannel 1 and assumes a stretched, li ⁇ -linear shape. This is followed by electrical cutting of the DNA in a region of the nanochannel 1 in which the DNA is in the linear form.
  • the pulse duration is 100 ys.
  • the pulse is applied at a predetermined frequency of 1 Hz to the DNA in the nanochannel 1.
  • Each pulse leads to a double strand break in the DNA, so that each one
  • double-stranded DNA fragment is obtained. Due to the con ⁇ constant rate at which the DNA is rinsed through the nanochannel 1, the DNA fragments have an equal length.
  • the DNA fragments are each 200 base pairs in length and are obtained from the nano channel 1 in which they are purged at the second end 5 of the nanochannel 1 of the nanochannel 1 ⁇ the.
  • the DNA fragments can be used for sequencing due to their uniform length.

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Analytical Chemistry (AREA)
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  • Wood Science & Technology (AREA)
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  • Physics & Mathematics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Biochemistry (AREA)
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  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne un procédé et un kit pour extraire au moins un fragment d'acide nucléique bicaténaire (9). Le procédé comprend la préparation d'au moins un nanocanal (1) chargé avec un seul acide nucléique bicaténaire (7), une coupe électrique de l'acide nucléique (7) pour produire un fragment bicaténaire afin d'obtenir au moins un fragment d'acide nucléique bicaténaire (9), et l'extraction du fragment d'acide nucléique (9) à partir du nanocanal (1).
PCT/EP2014/055562 2013-04-15 2014-03-20 Procédé d'extraction d'au moins un fragment d'acide nucléique bicaténaire Ceased WO2014170084A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE102013206657.0 2013-04-15
DE102013206657.0A DE102013206657A1 (de) 2013-04-15 2013-04-15 Verfahren zum Gewinnen von mindestens einem doppelsträngigen Nukleinsäure-Fragment

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WO2014170084A1 true WO2014170084A1 (fr) 2014-10-23

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PCT/EP2014/055562 Ceased WO2014170084A1 (fr) 2013-04-15 2014-03-20 Procédé d'extraction d'au moins un fragment d'acide nucléique bicaténaire

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DE (1) DE102013206657A1 (fr)
WO (1) WO2014170084A1 (fr)

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010111605A2 (fr) * 2009-03-27 2010-09-30 Nabsys, Inc. La présente invention concerne des dispositifs et des procédés d'analyse de biomolécules et des sondes liées à celles-ci
WO2011097028A1 (fr) * 2010-02-08 2011-08-11 Genia Technologies, Inc. Systèmes et procédés permettant de manipuler une molécule dans un nanopore
US20110224098A1 (en) * 2010-03-15 2011-09-15 International Business Machines Corporation Nanopore Based Device for Cutting Long DNA Molecules into Fragments
US20110308949A1 (en) * 2010-06-22 2011-12-22 International Business Machines Corporation Nano-fluidic field effective device to control dna transport through the same

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010111605A2 (fr) * 2009-03-27 2010-09-30 Nabsys, Inc. La présente invention concerne des dispositifs et des procédés d'analyse de biomolécules et des sondes liées à celles-ci
WO2011097028A1 (fr) * 2010-02-08 2011-08-11 Genia Technologies, Inc. Systèmes et procédés permettant de manipuler une molécule dans un nanopore
US20110224098A1 (en) * 2010-03-15 2011-09-15 International Business Machines Corporation Nanopore Based Device for Cutting Long DNA Molecules into Fragments
US20110308949A1 (en) * 2010-06-22 2011-12-22 International Business Machines Corporation Nano-fluidic field effective device to control dna transport through the same

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
MAKUSU TSUTSUI ET AL: "Transverse electric field dragging of DNA in a nanochannel", SCIENTIFIC REPORTS, vol. 2, 3 May 2012 (2012-05-03), XP055118806, DOI: 10.1038/srep00394 *

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