KR101347104B1 - Method and apparatus for transfering and extending a nucleic acid using immobilization of nanoparticles in a nanochannel - Google Patents

Abstract

The present invention comprises the steps of binding the nanoparticles to one end of the nucleic acid; Introducing the nucleic acid into the nanochannel; And a nucleic acid sequencing method comprising the step of fixing the nanoparticles in the singular or plural nanochannels; Single or plural nanochannels into which the nucleic acid is introduced; And a solution flowing within the nanochannel. The present invention can increase the spreading effect of the nucleic acid in the nanochannel, and because the solution containing the enzyme and the base flows, the accuracy of sequencing analysis is high, the noise signal is small, it is possible to detect the signal with high efficiency. In addition, even if the existing enzyme reaches the end of life, the polymerization reaction can be continuously caused by the new enzyme supplied from the flow solution, there is no limit to the length of the nucleic acid sequence analysis.

Description

TECHNICAL AND APPARATUS FOR TRANSFERING AND EXTENDING A NUCLEIC ACID USING IMMOBILIZATION OF NANOPARTICLES IN A NANOCHANNEL}

The present invention relates to methods and apparatus for nucleic acid sequencing.

There is a Sanger method as a nucleic acid analysis method, which takes a lot of time and money. In addition, multiple PCR (Polymerase Chain Reaction) and Sanger method have to be repeated to detect single nucleotide polymorphism (SNP).

In addition, the DNA molecules were randomly cut and then hybridized with specific fragments to identify sequences, and a sequencing method of hybridization, in which a number of overlapping sequences were connected by a computer, and a specific fragment was attached to a DNA molecule. However, there are methods such as massively parallel sequencing with stepwise enzymatic ligation and cleavage, which find out the sequence, but have the disadvantage of failing to analyze long sequences (Korean Patent Publication No. 10). -2006-0030835).

In addition, with the development of nano-structure processing technology and nano-optics, such as nanopore (nanopore), nanochannel (nanochannel), such as using a small size of the structure compared to the DNA base from each DNA There is a method of reading the order of electrical or optically. This technique has a problem of efficiently unfolding the tangled DNA in the nanostructures, and the problem of inferior accuracy in sequencing for long length DNA.

Korean Patent Publication No. 10-2006-0030835

The present invention provides a nucleic acid sequencing method and nucleic acid sequencing apparatus capable of analyzing a long length nucleic acid sequence by fixing and extending one end of the nucleic acid to continuously supply the nucleic acid synthase and base to solve the above problems do.

In order to achieve the above object, the present invention comprises the steps of combining the nanoparticles with one end of the nucleic acid; Introducing the nucleic acid into a single or a plurality of nanochannels; And it provides a nucleic acid sequence analysis method comprising the step of fixing the nanoparticles in the nanochannel.

In the nucleic acid sequencing method, the step of introducing into the nanochannel may be extended at the same time as the nucleic acid to which the nanoparticles are bound in the channel, and may be further extended by adding the nucleic acid after the step of immobilizing the nanoparticles.

After fixing in the nucleic acid sequencing method, it provides a nucleic acid sequencing method further comprising the step of synthesizing the nucleic acid by introducing a solution containing the nucleic acid synthetase and the base into the nanochannel.

In the nucleic acid sequencing method, the nanoparticles may be selected from the group consisting of proteins, aptamers, gold nanoparticles, and mixtures thereof.

In the nucleic acid sequence analysis method, the diameter of the nanoparticles may be 2 to 100 times the diameter of the nucleic acid.

In the nucleic acid sequencing method, the nanoparticles may be combined with the ends of the nucleic acid by mixing at a ratio of 10 to 10,000 times the number of nucleic acids.

In the nucleic acid sequence analysis method, the diameter of the nanochannel may be 10 to 100 nm.

In the immobilizing step of the nucleic acid sequencing method, the immobilization of the nanoparticles or the extension of the nucleic acid may be performed by genophoresis, light capture at a singular or plural point, formation of a singular or plural protrusions in the nanochannel, or pH adjustment. have.

In the nucleic acid sequencing method, the nucleic acid synthase may include a nucleic acid polymerase.

In the nucleic acid sequencing method, the base may be labeled with an antibody, oligopeptide, radioisotope, or fluorescence.

In the nucleic acid sequence analysis method, the solution may be flowing at a rate of 100-10000 times the nucleic acid sequence polymerization rate in the nanochannel, and the flow rate may be adjusted according to the conditions of polymerization and signal detection of the nucleic acid.

Synthesizing the nucleic acid in the nucleic acid sequence analysis method may include analyzing a sequence by detecting a signal of a base to be synthesized.

In the nucleic acid sequencing method, the nucleic acid may be single stranded DNA or single stranded RNA.

The present invention is a nucleic acid having a nanoparticle attached to one end; Single or plural nanochannels into which the nucleic acid is introduced; And a solution flowing inside the nanochannel, wherein the solution includes a nucleic acid synthetase and a base.

The nucleic acid sequencing device may further include a device for fixing the nanoparticles or extending the nucleic acid.

The nucleic acid sequencing device may further include a device for detecting nucleic acid synthesis.

In the nucleic acid sequencing device, the nanoparticles may be selected from the group consisting of proteins, aptamers, gold nanoparticles, and mixtures thereof.

In the nucleic acid sequencing device, the diameter of the nanochannel may be 10 to 100 nm.

In the nucleic acid sequencing device, the nucleic acid may be single stranded DNA or single stranded RNA.

The present invention can enhance the unfolding effect of nucleic acid in the nanochannel, and since the signal by the base synthesized to the nucleic acid in the channel is detected at a single molecule level, the accuracy of sequencing analysis is high, and the noise signal is small and the signal is highly efficient. Can be detected. In addition, even if the existing enzyme reaches the end of life, the polymerization reaction can be continuously caused by the new enzyme supplied from the flow solution, there is no limit to the length of the nucleic acid sequence analysis.

FIG. 1 shows immobilization of nanoparticles within nanochannels and stretching tangled single stranded DNA (ssDNA).
2 shows the synthesis of nucleic acids in nanochannels.
Figure 3 shows the signal detection process by nucleic acid synthesis.
Figure 4 shows an embodiment showing a nucleic acid to which nanoparticles of various sizes are bound.
Figure 5 shows an embodiment showing a process in which the nucleic acid to which the nanoparticles are bound is introduced into the nanochannel to cause the primary unfolding.
6 illustrates an embodiment of fixing nanoparticles in a nanochannel.
Figure 7 shows an embodiment of the enzyme synthesis in the nanochannel.
Figure 8 shows an embodiment showing the difference in speed between the base polymerized on the nucleic acid strand and the floating in the channel when detecting the fluorescent signal.

 Nucleic acids such as DNA or RNA are present in a twisted state in the form of a thread, and if passed through the nanochannels, some unfolding effect can be obtained, but there are limitations. The present inventors have developed a method for fixing one end of a nucleic acid in order to maximize the spreading effect of the nucleic acid.

Hereinafter, the present invention will be described in detail.

Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

In the present invention, the nucleic acid refers to single-stranded DNA (deoxyribonucleic acid) or single-stranded RNA (ribonucleic acid), genomic DNA, plasmid DNA, phage DNA, recombinant DNA, mRNA, rRNA, tRNA, recombinant RNA, microRNA, etc. It includes.

One embodiment of the present invention comprises the steps of combining the nanoparticles with one end of the nucleic acid; Introducing the nucleic acid into a single or a plurality of nanochannels; And it provides a nucleic acid sequence analysis method comprising the step of fixing the nanoparticles in the nanochannel.

In one embodiment of the invention the nucleic acid may be single stranded DNA or single stranded RNA.

In one embodiment of the present invention, the nanoparticles may be selected from the group consisting of proteins, aptamers, nanoparticles of various materials, and mixtures thereof.

The diameter of the nanoparticles is preferably larger than the diameter of the nucleic acid strand to effectively fix in the channel, or less than the diameter of the channel to be introduced into the nanochannel. Therefore, it may be 2 to 100 times the nucleic acid diameter, specifically 5 to 50 times. If less than 2 times difficult to fix the nanoparticles, more than 100 times may be difficult to introduce into the nanochannel. Figure 4 shows an embodiment showing a nucleic acid to which nanoparticles of various sizes are bound. (a) shows the case where the nanoparticle diameter is twice the diameter of the nucleic acid strand, (b) shows the case where the nanoparticle diameter is 5 to 50 times the diameter of the nucleic acid strand, and (c) the nanoparticle diameter is the nucleic acid strand It shows the case 100 times the diameter.

In one embodiment of the present invention, the nanoparticles may be mixed with the nucleic acid at a ratio of 1 to 10000 times, specifically, 10 to 1000 times the number of nucleic acids, to bind to the ends of the nucleic acids. If you mix more than 10000 times, the coupling efficiency may decrease.

The nanochannels may be singular or plural. Analyzing nucleic acid sequences using a plurality of nanochannels can simultaneously analyze nucleic acid sequences in parallel, thereby doubling the processing speed and minimizing errors due to repetitive sequence analysis.

In one embodiment of the present invention, the diameter of the nanochannel may be 10 to 100 nm, specifically, 30 to 70 nm. This takes into account the persistence length and Kuhn length of the nucleic acid. If it is less than 10 nm, it is difficult to introduce a DNA chain having a permanent length of 50 nm into the nanochannel. If it is more than 100nm, it is difficult to fix the nanoparticles, and the spreading efficiency of nucleic acids will be reduced. In the case of single-stranded DNA analysis, considering that the endurance length is 5 nm or less, it is desirable to minimize the diameter of the nanochannel at a level that can be introduced.

There is no limitation on the length of the nanochannels. All that is needed is a size larger than the length of the desired nucleic acid.

In the process of introducing the nucleic acid bound to the nanoparticles in the nanochannel, the aggregated nucleic acids may be unfolded. At this time, the nucleic acid ends to which the nanoparticles are bound move slowly due to the mass asymmetry of the nanoparticles and the nucleic acid, and the other end to which the nanoparticles are not bound moves relatively fast in the channel direction. This differential migration can lead to unfolding of the primary nucleic acid. Figure 5 is an embodiment showing the process of the first unfolding is introduced by the nanoparticle-bound nucleic acid is introduced into the nanochannel, (a) shows that the nucleic acid is first unfolded by using mass asymmetry.

In addition, unfolding of nucleic acid may occur due to the effect of the electrical properties of the nanoparticles to be bound. For example, if the bound nanoparticles are electrically neutral, the electrophoretic movement in the electric field is slower than that of negatively charged nucleic acid strands, resulting in a rate difference. This is because it can lead to nucleic acid. Figure 5 (b) shows that the nucleic acid is first unfolded by using the charge asymmetry.

In one embodiment of the present invention, the fixing may be to stretch the nucleic acid simultaneously with or after fixing the nanoparticles. Stretching the nucleic acid means spreading the nucleic acid. Fixation of the nanoparticles or extension of the nucleic acid may be achieved by applying an electric field or adding an external force and the surrounding physicochemical environment. Specifically, it may be made by the electrophoresis, light capture, or pH control induced by the nanochannel protrusion.

For example, a dielectrophoretic force induced by electrophoresis on the nanochannel may be generated to fix the nanoparticles and unfold the nucleic acid. By forming protrusions in the nanochannels, the electric field can be locally concentrated to fix the nanoparticles by the electrophoretic force. The protrusion may be formed in singular or plural in the nanochannel. In the case where a plurality of protrusions are formed in the nanochannel, a plurality of nanoparticles bound to the nucleic acid are fixed in the nanochannel, thereby allowing nucleic acid sequence analysis in parallel and further improving accuracy. Figure 6 is an embodiment for fixing the nanoparticles in the nanochannel, (a) shows the fixing of the nanoparticles by using electrophoresis.

 In addition, the nanoparticles can be fixed by light capture. The nanoparticles may be fixed by light trapping at a single point or a plurality of points within the nanochannels. When the light capture is carried out at a plurality of points, a plurality of nanoparticles bound to the nucleic acid are fixed in the nanochannel, thereby allowing nucleic acid sequence analysis in parallel and further improving accuracy. Figure 6 (b) shows an embodiment of fixing the nanoparticles using light capture.

Nucleic acid can also be expanded by adjusting the pH value of the solution introduced into the channel. As the pH value of the solution introduced with the nucleic acid in the nanochannel increases temporarily or stepwise, the nucleic acid can be spread.

By the above method, the electrophoretic flow is continued in the state where the nanoparticle portion is stably fixed, thereby improving the unfolding effect of the nucleic acid to a level of Contour length or more.

One embodiment of the present invention may further include the step of synthesizing the nucleic acid by introducing a solution containing a nucleic acid synthetase and a base into the nanochannel after the fixing step. Figure 7 shows an embodiment of the enzyme synthesis in the nanochannel.

In one embodiment of the present invention the solution may further contain equivalents such as ladder-forming oligonucleotide primers and / or chimeric oligonucleotide primers, buffering components, magnesium salts and components necessary for carrying out the amplification reaction.

In one embodiment of the present invention, the nucleic acid synthase may include a nucleic acid polymerase. Any DNA polymerase having helix substitution activity can be used according to the present invention. Examples thereof include large fragments of DNA polymerase I (Klenow fragment) derived from Escherichia coli (E. coli), as well as Bacillus stiadermophilus (hereafter described as B. st) and Bacillus caldotenex DNA polymerases lacking 5 ′ → 3 ′ exonuclease activity derived from thermophilic bacteria of the genus Bacillus, hereinafter described as B. ca. Both mesophilic and heat-resistant DNA polymerases can be preferably used according to the present invention.

In one embodiment of the invention the base may contain dNTPs, modified deoxyribonucleotides or deoxynucleotide triphosphate analogs.

In one embodiment of the invention the base may be labeled with an antibody, oligopeptide, radioisotope or fluorescence.

In one embodiment of the present invention, the term “label” means a detectable compound or composition conjugated directly or indirectly to an antibody, oligopeptide or other organic molecule, and is itself a detectable or detectable matrix compound or composition. It can catalyze the chemical alteration of. In addition, examples of the label include a chemiluminescent label, a Fluorescence resonance energy transfer (FRET) label, a quantum dot label or a metal label.

In one embodiment of the present invention, the fluorescent label is, for example, Cy-5, Cy-3, Alexa 647, Alexa 488, organic fluorescent labels including TOTO, etc., biotin-binding material, tetramethyltamine (TMR) , Tetramethylhodamine isocyanate (TMRITC), x-rhodamine, Texas red and the like can be used. Other fluorescent labels that can be used in the present invention will be known to those of ordinary skill in the art.

In one embodiment of the present invention, the solution may be to flow at a constant speed in the nanochannel. For example, the flow rate of the solution may be 100-10000 times the rate of nucleic acid sequence polymerization. If it is less than 100 times the polymerization rate, since the movement speed of the base in a solution is slow, the base in a fluid solution acts as a noise of the fluorescent signal which detects a polymerization reaction. Therefore, the difference between the polymerization extension rate of the base participating in the polymerization reaction and the flow rate of the base in the solution not participating in the polymerization is inferior, resulting in poor signal analysis accuracy. On the other hand, when it is more than 10000 times the polymerization rate, the efficiency of the polymerization reaction itself may be lowered.

The flow rate of such a solution can be applied by varying with time. In order to stably polymerize the enzyme and base, the rate of the solution is set to 0 temporarily to induce the reaction, and then a constant flow rate is applied when detecting the fluorescent signal. This allows relatively stable synthesis and signal detection.

The flow of solution can utilize electrophoretic forces by placing electrodes at both ends of the channel. At this time, when the solution at the inlet of the nanochannel is mostly moved to the outlet, a solution of the same concentration can be supplied to the inlet to maintain a constant solution concentration, flow rate, and environment. Figure 7 (a) shows an embodiment of flowing the solution using the electrophoretic force, enzyme synthesis of nucleic acid.

In addition, the inlet and outlet of the nanochannels can be connected to each other to pump the solution. At this time, there is an advantage that can maintain the flow rate of the solution, it is efficient because there is an advantage that can be utilized by circulating the enzyme or base that has not reacted. 7 (b) shows an embodiment in which the inlet and the outlet of the nanochannels are connected to each other to pump the solution, circulate the solution, and enzymatically synthesize the nucleic acid.

In one embodiment of the present invention the step of synthesizing the nucleic acid comprises analyzing the sequence by detecting a signal of the base to be synthesized.

In the present invention, the nucleic acid synthase induces a polymerization reaction between the nucleic acid and the base. Since the nucleic acid synthetase and the bases are continuously supplied by the circulation of the solution in the channel around the nucleic acid, even if one enzyme is inactivated during the polymerization, the polymerization may be continuously performed by other newly supplied enzymes. If the nucleic acid polymerase is not continuously supplied and is a single enzyme, the polymerase is likely to be inactivated by prolonged exposure to a high energy laser for fluorescence detection. In this case, there is a problem in that the length of nucleic acid synthesis capable of detecting a fluorescent signal is limited. However, the present invention is not limited to the length of nucleic acid that can be sequenced because the nucleic acid synthetase is continuously supplied.

In addition, the concentration of the nucleic acid synthesis base in the solution can be reacted with only a low concentration compared to the conventional sequencing method based on enzyme synthesis. In the conventional method using a nanostructure, since the base reaches the enzyme by diffusion in the solution, a solution containing a base having a concentration corresponding to several tens of Km (the concentration at which the reaction occurs) was used. On the other hand, in the case of the present invention, since the solution containing the base in the nanochannel is directly applied to the enzyme by flow, it can be reacted at a concentration corresponding to the Km value only.

In the present invention, the sequence analysis detects the base signal to be synthesized, and the sequence flowing in the channel has a relatively short residence time in the detection space by the laser due to flow and diffusion motion in the channel. On the other hand, the base attached to the nucleic acid sequence compared to the bases in the surrounding fluid solution has a very long residence time at a certain position for emitting a fluorescent signal, so that the fluorescence is stable and strong, thereby increasing the efficiency of detecting the fluorescent signal. This has the advantage of significantly reducing the noise signal caused by the surrounding fluorescent base than the prior art. Figure 8 shows an embodiment showing the difference in speed between the base polymerized on the nucleic acid strand and the floating in the channel when detecting the fluorescent signal. In the present embodiment, the residence time in the detection space of the base to be polymerized is about 10-30 msec, and the residence time in the detection space of the base suspended in the channel is about 10 sec-α. Where α is due to the force generated by the flow of the solution. According to the present invention, since the retention time of the polymerization base is 1000 times or more different than the retention time of the floating base, there is an effect that the efficiency of signal detection can be improved at a lower concentration than the conventional one.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Claims (19)

Binding the nanoparticles to one end of the nucleic acid;
Introducing the nucleic acid into a single or a plurality of nanochannels; And
Fixing and extending the nucleic acid in the nanochannel comprising the step of fixing the nanoparticles in the nanochannel. The method of claim 1,
Wherein said immobilizing comprises elongating the nucleic acid simultaneously with or after immobilization of the nanoparticles. The method of claim 1,
After the fixing step,
A method of transporting and stretching nucleic acids in a nanochannel, further comprising the step of synthesizing the nucleic acid by introducing a solution comprising a nucleic acid synthetase and a base into the nanochannel. The method of claim 1,
The nanoparticles are selected from the group consisting of proteins, aptamers, gold nanoparticles and mixtures thereof. 5. The method of claim 4,
The diameter of the nanoparticles is 2 to 100 times the diameter of the nucleic acid, characterized in that the transfer and extension method of the nucleic acid in the nanochannel. The method of claim 1,
Said nanoparticles are mixed at a ratio of 10 to 10,000 times the number of nucleic acids to bind to the ends of the nucleic acids, characterized in that the transport and extension of nucleic acids in the nanochannel. The method of claim 1,
The nanochannel diameter is 10 ~ 100nm, characterized in that the transfer and extension method of the nucleic acid in the nanochannel. 3. The method of claim 2,
In the fixation step, the fixation of the nanoparticles or the elongation of the nucleic acid is performed by genophoresis, light capture at the singular or plural points, formation of the singular or plural protrusions in the nanochannel, or pH adjustment. Of nucleic acid transport and elongation. The method of claim 3,
The nucleic acid synthetase comprises a nucleic acid polymerase method of transferring and stretching the nucleic acid in the nanochannel. The method of claim 3,
Wherein said base is labeled with an antibody, oligopeptide, radioisotope or fluorescence. The method of claim 3,
The solution is a method for transporting and stretching nucleic acids in the nanochannel, characterized in that the flow at a constant rate of 100-10000 times the rate of polymerization of the nucleic acid sequence in the nanochannel. The method of claim 3,
Synthesizing the nucleic acid comprises analyzing a sequence by detecting a signal of a base to be synthesized. 13. The method according to any one of claims 1 to 12,
Wherein said nucleic acid is single-stranded DNA or single-stranded RNA. Nucleic acids having nanoparticles attached to one end thereof;
Single or plural nanochannels into which the nucleic acid is introduced; And
It includes a solution flowing in the nanochannel,
The solution comprises a nucleic acid synthetase and a base, characterized in that for transporting and stretching the nucleic acid in the nanochannel. 15. The method of claim 14,
The device further comprises a device for immobilizing or stretching the nucleic acid nanoparticles device for transporting and stretching the nucleic acid in the nanochannel. 15. The method of claim 14,
Wherein said device further comprises a device for detecting nucleic acid synthesis. 15. The method of claim 14,
The nanoparticle is a protein, aptamers, gold nanoparticles and mixtures thereof, characterized in that the transport and stretching apparatus of nucleic acid in the nanochannels, characterized in that characterized in that the group. 15. The method of claim 14,
A device for transferring and stretching nucleic acids in a nanochannel, characterized in that the diameter of the nanochannel is 10 ~ 100nm. 19. The method according to any one of claims 14 to 18,
Wherein said nucleic acid is single-stranded DNA or single-stranded RNA.

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