WO2012050353A2 - Method whereby the base sequence of a target polynucleotide is non-specifically extended - Google Patents

Abstract

The present invention relates to a method whereby the base-sequence of a target polynucleotide is non-specifically extended to the base sequence length required for an intended purpose. The present invention can improve the sensitivity of detection or the reproducibility of detection of target polynucleotides, and can be put to use as a basic technique that can be put to service in various technologies for analysing single nucleotide polymorphism (SNP) and whether a target polynucleotide is present in a sample by using electrical-signal based biosensors and microfluidic chips and PCR amplification products; for example various technologies such as PCR-SSCP, microarray chips, multiplex PCR, real-time PCR, RFLP and electrophoretic analysis.

Description

Nonspecifically extending the base sequence of the target polynucleotide

The present invention relates to a method for nonspecifically extending the base sequence of a target polynucleotide, a primer composition for the same, and a kit for improving the sensitivity of the target polynucleotide detection, and more particularly, in detecting the presence of a target polynucleotide in a sample. The present invention relates to a method for non-specifically extending the nucleotide sequence of a target polynucleotide by the length of a nucleotide sequence necessary to improve the detection sensitivity or the reproducibility of a target polynucleotide, a primer composition for this, and a kit for improving the detection sensitivity of the target polynucleotide.

In addition, the present invention non-specifically extend the base sequence of the target polynucleotide according to the purpose, and improved detection sensitivity or detection reproducibility for the presence of the target polynucleotide by using the improved electrical property change of the extended target polynucleotide It relates to an electrical detection method.

In addition, the present invention non-specifically extend the base sequence of the target polynucleotide according to the purpose, by using the enhanced detection sensitivity of the extended target polynucleotide and / or signal enhancement of the labeling material detection sensitivity to the presence of the target polynucleotide Or a detection method with improved detection reproducibility.

In addition, the present invention can obtain an improved detection sensitivity for the target polynucleotide by non-specifically extending the base sequence of the target polynucleotide to be detected even when performing multiplex PCR on a plurality of gene templates. The present invention relates to a detection method that can be very useful for genotyping and SNP analysis of specific genes with alleles of dogs.

It has been suggested that the human genome map can be completed to make it more extensive to measure risks for disease, diagnose or predict disease, and predict response to drugs. Are revealed.

Depending on the function of the various genes, for example, by analyzing the mutation of a specific gene of a specific person can predict the degree of risk for the disease can be induced to prevent the disease in advance. In addition, genetic variation analysis of other infectious agents, such as viruses, that cause infections in living organisms, may be used to analyze whether the virus is resistant to a particular therapeutic drug, and the genotyping may be used to identify specific infectious agents. By predicting the response to a therapeutic drug, for example resistance, the patient can develop a personalized treatment.

However, in order to clarify the relationship between genetic variation and disease in humans and humans, it is necessary not only to analyze a large number of sequence information, but also to accurately and effectively satisfy various specificities and sensitivity while satisfying both specificity and sensitivity. You should be able to analyze it.

Generally, DNA sequencing to be searched for molecular genetic analysis or clinical diagnosis is amplified by polymerase chain reaction method using DNA polymerase (hereinafter referred to as "PCR"), and then its presence is determined in the sample. Done. Various methods have been developed using the PCR amplification technique to detect whether a DNA sequence to be found in a sample, that is, whether a target polynucleotide exists.

For example, PCR-SSCP (Orita, M. et. Al.), Which performs PCR amplification of a target polynucleotide to be detected, separates amplified sequences into respective chains, and then performs electrophoresis on a polyacrylamide gel. Genomics, 1989, 5: 8874-8879), a probe prepared in advance for a target polynucleotide was prepared in advance, and a DNA chip was prepared by using a microarray device on a glass slide. After amplification using a labeled specific primer, the amplification product is hybridized to a DNA chip, and a fluorescent signal is analyzed by a scanner.

In addition, recently, oligonucleotides such as primers or probes attached with thiol groups are fixed to the metal electrode surface using the SAM (Self Assembled Method) method, and then electrical changes after PCR amplification reaction or hybridization reaction are performed in the microfluidics chip. Various methods for detecting the presence of a target polynucleotide in a sample have been developed (see Korean Patent Publication No. 10-2004-0042021, Korean Patent Registration No. 10-0590546, etc.).

Therefore, it is important to improve the detection sensitivity of the target polynucleotide in order to detect the sensitive and accurate presence of the target polynucleotide in the sample, and the primer used for amplification of the target polynucleotide and the PCR amplified by the primer It can be said that the amplification product has a great influence on the detection sensitivity and reproducibility.

In order to ensure accurate detection and reproducibility of the target polynucleotide, it is advantageous that the single strand of the PCR amplification product is as long as possible. Since there is a problem that false-positive products are generated, the length of PCR amplification products is limited by the combination of specific forward and reverse primers.

For example, in microfluidics chips manufactured using MEMS (Micro-Electro-Mechanical-System) technology, PCR amplification reactions or hybridization reactions are carried out using primers or probes fixed on the metal electrode surface, The presence of a target polynucleotide in the sample is determined by detecting electrical change, ie impedance or capacitance change, with increasing or hybridization. However, the target polynucleotide bound to the primer or probe immobilized on the metal electrode surface does not have a sufficient length due to the design limitation of the specific primer in the amplification process, and thus has low electrical detection sensitivity and low reproducibility after PCR amplification reaction or hybridization reaction. There was a problem. In order to solve this problem, a sensor with an amplification circuit for amplification of CMOS and electrical detection values has been fabricated, but the manufacturing process is complicated and the manufacturing cost increases to commercialize, as well as the signal value of the positive amplification product. There was a problem that the noise value was also amplified together.

In order to solve this problem, Korean Patent No. 10-0969667 discloses a method of improving the detection sensitivity of PCR reaction or hybridization reaction and the presence of specific DNA sequences using a reference solution having a large dielectric constant. , Korean Patent No. 10-0969671 discloses a high-sensitivity biosensor in which a biocompatible dielectric thin film is formed on a sensing metal electrode surface in order to solve the PCR amplification interference and a decrease in detection sensitivity caused by nonspecific adsorption of proteins on the sensing metal electrode surface. It is starting.

However, these prior arts focus on the problem of reproducibility of detection by buffers and the problem of nonspecific adsorption to sensing metal electrodes, which extends the target polynucleotide in a nonspecific manner by the desired length, thereby providing sensitivity to electrical, optical or electrochemical detection. And not intended to improve reproducibility.

In addition, the above-described DNA chip also has a problem that it is difficult to accurately analyze the fluorescence signal if the length of the nucleotide sequence of the PCR amplification product is short due to the effect of the position of label (POL) caused by the spatial problem.

Accordingly, in the art to which the present invention belongs, non-specifically extending the base sequence of the target polynucleotide by the base sequence length necessary to improve the detection sensitivity or detection reproducibility of the target polynucleotide in detecting the presence of the target polynucleotide in the sample. It can be said that the provision of a new method is required.

The present invention relates to a method for nonspecifically extending the base sequence of a target polynucleotide, a primer composition for the same, and a kit for improving the sensitivity of the target polynucleotide detection, and more particularly, in detecting the presence of a target polynucleotide in a sample. It is an object of the present invention to provide a method for non-specifically extending the base sequence of a target polynucleotide by a base sequence length necessary to improve the detection sensitivity or detection reproducibility of the target polynucleotide, a primer composition, and a kit for improving the detection sensitivity of the target polynucleotide. .

It is also an object of the present invention to non-specifically extend the base sequence of the target polynucleotide according to the purpose, and to improve the detection sensitivity or detection reproducibility of the presence of the target polynucleotide by using the improved electrical property change of the extended target polynucleotide. It is to provide an improved electrical detection method.

It is also an object of the present invention to non-specifically extend the base sequence of the target polynucleotide according to the purpose, and to improve the detection sensitivity of the extended target polynucleotide and / or signal enhancement of the labeling material to determine the presence of the target polynucleotide. A detection method having improved detection sensitivity or detection reproducibility is provided.

In addition, an object of the present invention is to obtain an improved detection sensitivity for the target polynucleotide by non-specifically extending the base sequence of the target polynucleotide to be detected even when performing multiplex PCR on a plurality of gene templates In addition, the present invention provides a detection method that can be very useful in genotyping and SNP analysis of a specific gene having a large number of allele types.

As a result of solving the above technical problem and intensive studies to meet the object of the present invention, the present inventors do not have homology with the nucleotide sequence of the genomic gene at the end of the first primer specifically binding to the target polynucleotide. A modified first primer that adds a first non-homologous sequence (hereinafter also referred to as a "zip code sequence") that does not complementally hybridize with a genomic gene as a non-combined nucleotide sequence, and target poly A second non-homologous sequence complementary to the first non-homologous sequence at the end of a second primer that specifically binds an extension polynucleotide that does not complementally hybridize with the nucleotide [hereinafter referred to as “second zip code Base sequence of the target polynucleotide when designing and using a modified second primer with the addition of a "zip code sequence" The present invention has been completed by focusing on the nonspecific extension and synthesis according to the present invention.

On the other hand, the method of non-specifically extending the base sequence of the target polynucleotide using the modified first primer and the modified second primer of the present invention biosensors and microfluid using the electrical signal described as an example in the embodiment of the present invention Various techniques for analyzing the presence and presence of target polynucleotides and single nucleotide polymorphisms (SNPs) in a sample using PCR amplification products, but not for Dix chips only, for example PCR-SSCP, microarray chips, multiplex PCR It should be understood as a fundamental technology that can be applied to technologies such as real-time PCR, RFLP, and electrophoresis techniques.

First, the terms used in the specification of the present invention will be described.

As used herein, a "target polynucleotide" is a single- or double-stranded DNA or RNA that is to be detected in a sample, and has a polynucleotide having a specific nucleotide sequence portion used for monobasic polymorphism (SNP) or genotyping. Nucleotides.

As used herein, "extending polynucleotide" does not hybridize complementarily with "target polynucleotide" and nonspecifically "target polynucleotide" for improved detection sensitivity or reproducibility in the presence or absence of "target polynucleotide". By polynucleotide used to extend. For example, a DNA template included in a commercially available kit for PCR amplification can be used as an "extension polynucleotide."

As used herein, the "modified first primer" is a nucleotide sequence that does not have homology with the nucleotide sequence of the genomic gene at the end of the first primer specifically binding to the target polynucleotide, and hybridizes complementarily with the genomic gene. It refers to a primer to which the first non-homologous sequence (hereinafter, also referred to as "first zip code sequence") which is not added. A "modified first primer" can include a forward modified first primer that extends a target polynucleotide in a downstream direction, and a reverse modified first primer that extends a target polynucleotide in an upstream direction.

As used herein, a "modified second primer" refers to a first non-homologousity of the modified first primer at the end of a second primer that specifically binds to an extension polynucleotide that does not complementally hybridize with the target polynucleotide. It means a primer added with a second non-homologous base sequence (hereinafter also referred to as "zip code sequence") complementary to the base sequence. "Modified second primer" may include a forward modified second primer that extends the extending polynucleotide in the downstream direction, and a reverse modified second primer that extends the extending polynucleotide in the upstream direction.

When designing a modified first primer and a modified second primer according to the present invention, the first zip code sequence of the forward modified first primer is complementary in reverse (i.e., the second zip code sequence of the reverse modified second primer) , Alternately complementary to each other based on the 5'-3 'direction, wherein the first zip code sequence of the reverse modified first primer is complementary in reverse (ie, complementary to the second zip code sequence of the forward modified second primer). , Complementary to each other based on the 5'-3 'direction (see FIG. 1).

Method for non-specifically extending the base sequence of the target polynucleotide of one embodiment of the present invention,

(a) a first non-homologous sequence not complementarily hybridized with the genomic gene as a base sequence having no homology with the nucleotide sequence of the genomic gene at the end of the first primer specifically binding to the target polynucleotide, or complementary thereto Preparing a modified first primer added with a specific sequence,

(b) preparing an extension polynucleotide that is not complementarily hybridized with the target polynucleotide and is used to nonspecifically extend the target polynucleotide;

(c) a nucleotide sequence having no homology with the nucleotide sequence of the genomic gene at the end of the second primer that specifically binds to the extension polynucleotide, and is not complementarily hybridized with the genomic gene, but not the hybridization of the modified first primer. 1 preparing a second non-homologous sequence complementary to the non-homologous sequence or a modified second primer added with a sequence complementary thereto;

(d) using the modified first primer to add the first non-homologous or complementary sequence to the end of the target polynucleotide to obtain an extended target polynucleotide, and using the modified second primer. Adding a second non-homologous sequence or a base sequence complementary thereto to obtain an extension polynucleotide for extension;

(e) the first non-homologous sequence added to the end of the target polynucleotide or the base complementary thereto, and the second non-homologous sequence added to the extension polynucleotide end or the sequence complementary thereto complement Annealing to an enemy to extend the target polynucleotide and the extension polynucleotide obtained in step (d) with respect to each other.

In addition, in the method of non-specifically extending the base sequence of the target polynucleotide of one embodiment of the present invention, the target polynucleotide obtained in the step (d) may be immobilized on the solid phase surface.

According to the method of the present invention, the DNA of each of the target polynucleotide and the extension polynucleotide by annealing (Annealing) while the ends of the target polynucleotide end and the extension polynucleotide obtained in step (d) are complementary to each other. The DNA backbone functions as a primer of a PCR amplification reaction with respect to each other, and a dual-extension is performed extending in both directions about the base sequences staggered to each other. As a result, a target polynucleotide is produced that is extended and amplified in a nonspecific manner by the extending polynucleotide.

In the method for non-specifically extending the base sequence of the target polynucleotide of an embodiment of the present invention, the modified first primer is a forward modification by adding one first non-homologous sequence to the 5 'end of the forward primer The first primer or the reverse modified first primer modified by adding another first non-homologous sequence to the 5 'end of the reverse primer, or both can be used.

In the method of non-specifically extending the base sequence of the target polynucleotide of an embodiment of the present invention, the modified second primer is reverse to the first non-homologous sequence of the forward modified first primer at the 5 'end of the reverse primer A reverse second modified primer that is modified by adding a second non-homologous sequence complementary to the second primer, or a second non-complementary complementary to the first non-homologous sequence of the reverse modified first primer at the 5 'end of the forward primer A forward modified second primer or both of which may be modified by adding a homologous base sequence.

In the method for non-specifically extending the base sequence of the target polynucleotide of one embodiment of the present invention, the extension polynucleotide extended in step (d) is stored in a double chain form, or PCR amplification reaction using magnetic beads, etc. It can be stored and separated into single chains.

In the method of non-specifically extending the base sequence of the target polynucleotide of an embodiment of the present invention, a combination of the forward modified first primer and the reverse modified second primer, or the reverse modified first primer and the forward direction Depending on the selection of the combination of modified second primers of the target polynucleotide can be nonspecifically extended in only one direction. In addition, the extension direction of the target polynucleotide may be determined depending on whether the extension polynucleotide is used in ss-DNA form or ds-DNA form.

In the method for non-specifically extending the nucleotide sequence of the target polynucleotide of an embodiment of the present invention, the amplification polynucleotide having another sequence in addition to the extension polynucleotide extended in the step (d) is prepared by further amplification Can be used to further extend the target polynucleotide.

The method of non-specifically extending the base sequence of the target polynucleotide of one embodiment of the present invention may further include extending the target polynucleotide extended in the step (e) by using the extension polynucleotide. . In this case, the nucleotide sequence of the target polynucleotide can be extended in a nonspecific manner by a desired length.

The method of non-specifically extending the base sequence of the target polynucleotide of one embodiment of the present invention by non-specifically extending the base sequence of the target polynucleotide to be detected even when performing multiplex PCR on a plurality of gene templates Improved detection sensitivity for the target polynucleotide can be obtained. In addition, it can be very useful in genotyping and / or SNP analysis of a particular gene having a large number of allele types.

Method for non-specifically extending the base sequence of the target polynucleotide of another embodiment of the present invention,

(a) a first non-homologous sequence not complementarily hybridized with the genomic gene as a base sequence having no homology with the nucleotide sequence of the genomic gene at the end of the first primer specifically binding to the target polynucleotide, or complementary thereto Preparing a modified first primer added with a specific sequence,

(b) preparing an extension polynucleotide that is not complementarily hybridized with the target polynucleotide and is used to nonspecifically extend the target polynucleotide;

(c) a nucleotide sequence having no homology with the nucleotide sequence of the genomic gene at the end of the second primer that specifically binds to the extension polynucleotide, and is not complementarily hybridized with the genomic gene, but not the hybridization of the modified first primer. 1 preparing a second non-homologous sequence complementary to the non-homologous sequence or a modified second primer added with a sequence complementary thereto;

(d) using the modified first primer to add the first non-homologous or complementary sequence to the end of the target polynucleotide to obtain an extended target polynucleotide, and using the modified second primer. Adding a second non-homologous sequence or a base sequence complementary thereto to obtain an extension polynucleotide for extension;

(e) modifying a forward nucleotide oligomer complementary to the first non-homologous sequence or complementary to the target polynucleotide terminus with a functional group for particle attachment, and adding the agent added to the extension polynucleotide terminus. (2) modifying a reverse nucleotide oligomer complementary to a nonhomologous sequence or a sequence complementary thereto to a functional group for particle attachment,

(f) attaching the modified forward and reverse nucleotide oligomers to the particles,

(g) the forward nucleotide oligomer attached to the particle obtained in step (f) is extended by annealing complementarily with the first non-homologous sequence added to the target polynucleotide termini or a complementary nucleotide sequence, and the (f) The reverse nucleotide oligomer attached to the particle obtained in the step) includes the step of annealing and extending complementarily with a second non-homologous sequence added to the extension polynucleotide terminal or a sequence complementary thereto.

According to the method of the present invention, the target polynucleotide is linked to the polynucleotide for extension through the particles and has the effect of improving the detection sensitivity by extending the base sequence by the desired base sequence length in a non-specific manner.

The method for non-specifically extending the base sequence of the target polynucleotide of another embodiment of the present invention comprises a second non-homologous sequence in which the reverse nucleotide oligomer attached to the particle in step (g) is added to the extension polynucleotide terminal or The nucleotide sequence extended by annealing complementary to the complementary sequence may be further extended using the extension polynucleotide again. In this case, the target polynucleotide is linked with the polynucleotide for extension through the particle, and the base sequence can be extended by the desired length of the base sequence to improve detection sensitivity. In particular, detection sensitivity or reproducibility may be improved by adjusting the number of extension polynucleotides linked to the target polynucleotide according to the number of nucleotide oligomers attached to the particles.

In addition, in a method of non-specifically extending the base sequence of the target polynucleotide of another embodiment of the present invention, the reverse nucleotide oligomer attached to the particle further comprises an extension polynucleotide having a sequence other than the extension polynucleotide. After preparation by amplification, these additional extension polynucleotides can also be annealed to extend and amplify.

Primer composition for non-specifically extending the base sequence of the target polynucleotide of one embodiment of the present invention,

A first non-homologous sequence not complementarily hybridized with the genomic gene as a base sequence having no homology with the nucleotide sequence of the genomic gene at the end of the first primer specifically binding to the target polynucleotide, or a sequence complementary thereto And a modified first primer added with,

A base sequence that does not have homology with the nucleotide sequence of the genomic gene at the end of the second primer that specifically binds to the extending polynucleotide used to nonspecifically extend the base sequence of the target polynucleotide, complementarily with the genomic gene. And a modified second primer which does not hybridize and adds a second non-homologous sequence complementary to the first non-homologous sequence of the modified first primer or a sequence complementary thereto.

In the primer composition for non-specifically extending the base sequence of the target polynucleotide of one embodiment of the present invention, the modified first primer is a forward direction modified by adding one first non-homologous sequence to the 5 'end of the forward primer A modified first primer or a reverse modified first primer modified by adding another first non-homologous sequence to the 5 'end of a reverse primer, or both.

In the primer composition for non-specifically extending the base sequence of the target polynucleotide of one embodiment of the present invention, the modified second primer and the first non-homologous base sequence of the forward modified first primer at the 5 'end of the reverse primer A reverse modified second primer modified by adding a second non-homologous sequence complementary to the reverse direction, or a second complementary reverse to the first non-homologous sequence of the reverse modified first primer at the 5 'end of the forward primer Forward modified second primers modified with the addition of non-homologous sequences, or both.

Primer composition for non-specifically extending the base sequence of the target polynucleotide of one embodiment of the present invention, the combination of the forward modified first primer and the reverse modified second primer, or the reverse modified first primer and the It can be used in combination with a forward modified second primer.

In a primer composition for non-specifically extending the base sequence of a target polynucleotide of an embodiment of the present invention, the forward modified first primer specifically amplifies the target polynucleotide and a 5 'end is immobilized on a solid phase surface. It may be a primer.

For example, the forward modified first primer may be an oligonucleotide having a nucleotide sequence of SEQ ID NO: 1, and the reverse modified first primer may be an oligonucleotide having a nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 8 have. In addition, the forward modified second primer may be an oligonucleotide having a nucleotide sequence of SEQ ID NO: 4, the reverse modified second primer may be an oligonucleotide having a nucleotide sequence of SEQ ID NO: 5.

Target polynucleotide detection sensitivity improvement kit of one embodiment of the present invention,

DNA polymerase, dNTPs and buffers,

A first non-homologous sequence not complementarily hybridized with the genomic gene as a base sequence having no homology with the nucleotide sequence of the genomic gene at the end of the first primer specifically binding to the target polynucleotide, or a sequence complementary thereto And a modified first primer added with,

A base sequence that does not have homology with the nucleotide sequence of the genomic gene at the end of the second primer that specifically binds to the extending polynucleotide used to nonspecifically extend the base sequence of the target polynucleotide, complementarily with the genomic gene. A modified second primer which does not hybridize and adds a second non-homologous sequence complementary to the first non-homologous sequence of the modified first primer or a sequence complementary thereto;

An extension polynucleotide that is not complementarily hybridized with the target polynucleotide and is used to nonspecifically extend the target polynucleotide, wherein the second non-homologous sequence or the complementary sequence is added by the modified second primer. Extension polynucleotides.

In the target polynucleotide detection sensitivity improving kit of one embodiment of the present invention, the extension polynucleotide may be stored in a double chain form, or separated and stored in a single chain by a PCR amplification reaction using magnetic beads or the like. .

Target polynucleotide detection sensitivity improvement kit of one embodiment of the present invention is an extension polynucleotide having a sequence other than the extension polynucleotide is added to the second non-homologous sequence or a complementary sequence by the modified second primer. It may further include.

In the kit for improving detection sensitivity of an exemplary embodiment of the present invention, the modified first primer is a forward modified first primer modified by adding one first non-homologous sequence to the 5 'end of the forward primer, or A reverse modified first primer, or both, modified by adding another first non-homologous sequence to the 5 'end of the reverse primer.

In the target polynucleotide detection sensitivity improving kit of one embodiment of the present invention, the modified second primer is a second complementary reverse to the first non-homologous sequence of the forward modified first primer at the 5 'end of the reverse primer A reverse modified second primer modified by adding a non-homologous base sequence or a second non-homologous sequence complementary to the first non-homologous base sequence of the reverse modified first primer is added to the 5 'end of the forward primer. And a modified second primer in the forward direction, or both.

The target polynucleotide detection sensitivity improving kit of one embodiment of the present invention includes a combination of the forward modified first primer and the reverse modified second primer, or the reverse modified first primer and the forward modified second primer. It can be prepared in combination.

In the kit for detecting sensitivity of a target polynucleotide according to an embodiment of the present invention, the forward modified first primer may be a primer specifically amplifying the target polynucleotide and having a 5 'end fixed to a solid phase surface.

For example, the forward modified first primer may be an oligonucleotide having a nucleotide sequence of SEQ ID NO: 1, and the reverse modified first primer may be an oligonucleotide having a nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 8 have. In addition, the forward modified second primer may be an oligonucleotide having a nucleotide sequence of SEQ ID NO: 4, the reverse modified second primer may be an oligonucleotide having a nucleotide sequence of SEQ ID NO: 5.

The target polynucleotide detection sensitivity improving kit of one embodiment of the present invention comprises a forward nucleotide oligomer complementary to the first non-homologous sequence or a nucleotide sequence complementary thereto and the second non-homologous nucleotide sequence or a sequence complementary thereto. It may further comprise a particle to which the reverse nucleotide oligomer is attached.

Preferably, the forward nucleotide oligomer and the reverse nucleotide oligomer are attached to the particles after being modified with a functional group for particle attachment. It is further preferred that a plurality of forward nucleotide oligomers and reverse nucleotide oligomers be attached to the particles.

Optionally, the particles may be attached with additional nucleotide oligomers complementary to the extending polynucleotide having a sequence other than the extending polynucleotide.

Meanwhile, the particles used in the present invention may be gold particles, iron oxide nanoparticles, superparamagnetic nanoparticles, silica nanoparticles, magnetic beads or polymer beads containing iron oxide therein.

In addition, although not described in detail in the following examples, labeling a label, such as a fluorescent material, on the particles or the polynucleotide for extension, enhances the detection signal by the label and detects or detects reproducibility of the presence of a target polynucleotide. Can improve. It will be readily understood by those skilled in the art that labeling the labeling material on the particles or extension polynucleotides can be carried out using a variety of labeling methods known in the art. (Reference: MANJULA KURELLA et al., DNA Microarray Analysis of Complex Biologic Processes, 2001, Journal of the American Society of Nephrology 12: 1072-1078; Danh V. Nguyen et al., DNA Microarray Experiments: Biological and Technological Aspects, December 2002, BIOMETRICS 58: 701-717).

In this case, the labeling material is not limited to a specific fluorescent material, as well as a variety of fluorescent materials that can be identified as a fluorescent detector may be used, and enzymatic color reaction and isotopic labeling are also possible. For example, the label is Cy5, Cy3, biotin-binding material, EDANS (5- (2'-aminoethyl) amino-1-naphthalene sulfate), tetramethyltamine (TMR), tetramethyltamine isocyanate (TMRITC ), x-rhodamine or Texas red and the like, and it is preferable that the phosphor is Cy3 or Cy5. However, the present invention is not limited thereto, and various labeling materials such as radioactive materials or light emitting materials known in the art may be used.

According to the present invention, in detecting the presence of a target polynucleotide in a sample, the base sequence of the target polynucleotide can be non-specifically extended and amplified by the base sequence length necessary to improve detection sensitivity or detection reproducibility of the target polynucleotide. .

Therefore, according to the present invention, the base sequence of the target polynucleotide is non-specifically extended according to the purpose, and the detection sensitivity or the detection reproducibility of the presence of the target polynucleotide is improved by using the improved electrical property change of the extended target polynucleotide. You can.

In addition, according to the present invention, the base sequence of the target polynucleotide is non-specifically extended according to the purpose, and the detection of the presence of the target polynucleotide using the enhanced detection sensitivity of the extended target polynucleotide and / or the signal enhancement of the labeling substance. Sensitivity or detection reproducibility can be improved.

In addition, according to the present invention, even when performing multiplex PCR on a plurality of gene templates, non-specifically extending the base sequence of a target polynucleotide to be detected can obtain an improved detection sensitivity for the target polynucleotide, It can be very useful for genotyping and SNP analysis of specific genes with multiple allele types.

The above and other technical problems and features of the present invention will be apparent to those skilled in the art through the description of the embodiments of the present invention made with reference to the accompanying drawings.

1 illustrates a method of designing a combination of a forward modified first primer and a reverse modified second primer and a combination of a reverse modified first primer and a forward modified second primer according to an embodiment of the present invention. Drawing.

2 is a view for explaining a process of amplifying a target polynucleotide using a modified first primer in Example 1 of the present invention.

Figure 3 shows the nucleotide sequence of the target polynucleotide shown as an example of the target polynucleotide amplified according to the method of one embodiment of the present invention.

4 is a view for explaining a process of amplifying a polynucleotide for extension using a modified second primer in Example 1 of the present invention.

5 is a view for explaining a process of extending the base sequence of the amplified target polynucleotide using the extension polynucleotide amplified in Example 1 of the present invention.

FIG. 6 is amplified by extending a target polynucleotide in a fixed state on a solid phase using a forward primer immobilized on a solid phase and a modified first primer in a reverse direction in Example 2 of the present invention, and using a previously prepared extension polynucleotide. It is a figure explaining the process of extending the base sequence of a target polynucleotide by a desired length.

7 is a view illustrating a process of extending and amplifying a target polynucleotide in a fixed state on a solid phase by using a modified first primer in a reverse direction and a forward primer fixed on a solid phase in Example 3 of the present invention.

8 is linked to a target polynucleotide with an extended polynucleotide through a gold particle to which nucleotide oligomers modified with thiols are attached in Example 3 of the present invention, and a sequencing sequence is performed in a nonspecific manner to improve detection sensitivity. A diagram illustrating the process of extension and amplification.

9 is a diagram illustrating an amplified product amplified according to the process of FIG. 8.

FIG. 10 is PCR amplification of the target polynucleotide cytochrome CYP2C9 * 3 gene by dual extension using a reverse modified first primer, a forward modified second primer, and an extension polynucleotide. A diagram illustrating a process of extending the nucleotide sequence by a desired length. FP in FIG. 10 is a forward primer, RP is a reverse primer, Z2 and Z3 are zip code sequences that are complementary to each other in reverse (ie, complementary to each other based on the 5'-3 'direction). .

11 is a photograph (left) showing the results of gel electrophoresis after amplification of the cytochrome CYP2C9 * 3, CYP2C19 * 2 and CYP2C19 * 3 alleles using a single PCR method and multiplex PCR method of the related art; 10 is a photograph showing the results of gel electrophoresis after amplification using the multiplex PCR method by the dual extension method (dual extension) (right).

Hereinafter, the present invention will be described in more detail based on examples. The following examples of the present invention are not intended to limit or limit the scope of the present invention only to embody the present invention. From the detailed description and examples of the present invention, those skilled in the art to which the present invention pertains can easily be interpreted as belonging to the scope of the present invention. References cited in the present invention are incorporated herein by reference.

Example

Example 1: Extension of the nucleotide sequence of the target polynucleotide

Example 1-1 (Step 1): Extension of Target Polynucleotide Using Modified First Primer

1) A first non-homologous sequence which does not hybridize with the genomic DNA as a base sequence having no homology with the base sequence of the genomic DNA at the end of the first primer specifically binding to the target polynucleotide [hereinafter, " A modified first primer, which is a primer to which the first zip code sequence "is added, is prepared.

2) To this end, first, a target polynucleotide having a nucleotide sequence of SEQ ID NO: 3 as shown in FIG. 3 is prepared. Then, base sequences of the forward and reverse primers specific for the 5 'end and the 3' end of the target polynucleotide are determined, respectively. In addition, the first modified first primer (SEQ ID NO: 1) modified by adding one first zip code sequence (5'-CCCCAAACGTACCAAGCCCGCGTCG-3 ') to the 5' end of the forward primer (5'-GCAGCTCCAATGAGAACCTC-3 '). Or modified by changing another primer (SEQ ID NO: 5'-CGCGCGCAGCTGCAGCTTGCTCATG-3 ') by adding another first zip code sequence (5'-CGCGCGCAGCTGCAGCTTGCTCATG-3') to the 5 'end of the reverse primer (5'-AACGAAAAGGTTGGGGTCAT-3'). 2) is produced (see Fig. 2). In this embodiment, both of the forward modified first primer and the reverse modified first primer were prepared, but only primers in a desired direction of extension can be modified by adding a zip code sequence.

3) Meanwhile, the zip code sequence is a nucleotide oligomer having a sequence which is not in the nucleotide sequence of the genomic DNA and is a sequence designed not to hybridize non-specifically with the genomic DNA. It is noted that the zip code sequences used in the examples which follow are only mentioned for illustrative purposes and are not intended to limit the invention. Those skilled in the art will readily understand that the primers can be modified using various zip code sequences known in the art, in addition to the zip code sequences used in the examples described below (Ref. Slentz- Kesler et al., Polymorphism Analysis Using Single Base Chain Extension A Microsphere-Based, Genome Res., 2000, 10: 549-557).

The sequence of the modified first primer pair produced by modifying the zip code sequence is as follows. The underlined portion of the base sequence of the modified first primer pair below is a zip code sequence.

① the first modified primer in the forward direction:

5'- CCCCAAACGTACCAAGCCCGCGTCG GCAGCTCCAATGAGAACCTC-3 '(SEQ ID NO: 1)

② modified first primer in reverse direction:

5'- CGCGCGCAGCTGCAGCTTGCTCATG AACGAAAAGGTTGGGGTCAT-3 '(SEQ ID NO: 2)

4) 2X PCR master mix solution (i-StarMAX II) (purchased from Intron Biotechnology, Inc., Seongnam, Gyeonggi-do, Korea) 12 μl of target polynucleotide prepared in step 2) (SEQ ID NO: 3) 1 μl, and 2 μl of the modified first primer pair (SEQ ID NO: 1 and SEQ ID NO: 2) prepared in step 2) above, add 9 μl of secondary distilled water to adjust the total reaction volume to 24 μl. And the PCR reaction is carried out under the following conditions, and the temperature is maintained at 4 ℃ after the PCR reaction.

95 ℃ 5 minutes

95 ° C 30 seconds, 57 ° C 30 seconds, 72 ° C 30 seconds (30 cycles)

72 ° C. 5 minutes.

5) FIG. 2 schematically illustrates the PCR amplification process and the amplification product performed in step 4). When the target polynucleotide is amplified by the general forward and reverse primers, the PCR amplification product of the target polynucleotide is 407bp of base. On the other hand, when the target polynucleotide was amplified using the modified first primer pair of the present embodiment, the PCR amplification product of the target polynucleotide was the zip code sequence at the 5 'end and the 3' end and the sequence complementary thereto. This additional formation (407bp + 25bp + 25bp) will have a nucleotide sequence length of 457bp. At this time, the 5 'end and / or 3' end of the PCR amplification product of the target polynucleotide amplified using the modified first primer pair of the present embodiment any desired base sequence (in this embodiment, the first zip code sequence) Or a sequence complementary thereto) is further extended. This will be combined with the second zip code sequence of the modified second primer described below or a sequence complementary thereto.

Example 1-2 (Step 2): Extension of Extension Polynucleotide Using Modified Second Primer

1) a second non-homologous sequence complementary to the first non-homologous sequence of the modified first primer at the end of the second primer that specifically amplifies the extension polynucleotide that does not complementally hybridize with the target polynucleotide A modified second primer, which is a primer added with the following (hereinafter also referred to as a "zip code sequence"), is prepared.

2) In this embodiment, amplified DNA template (323 bp) of Promega's GoTaq ^® PCR Core System II (Promega, M7665) is prepared for extension polynucleotides, and such extension polynucleotides can be produced and stored in large quantities. . On the other hand, the extension polynucleotide is used to extend the length of the base sequence necessary to improve the detection sensitivity or detection reproducibility at the end of the PCR amplification product of the target polynucleotide obtained in step 1 of Example 1-1.

3) A primer base sequence provided by the manufacturer of the DNA template is used as a base sequence of a forward primer and a reverse primer specific for the 5 'end and the 3' end of the extension polynucleotide for the preparation of the modified second primer. The modified second primer (SEQ ID NO: 4) was modified by adding one second zip code sequence (5'-CATGAGCAAGCTGCAGCTGCGCGCG-3 ') to the 5' end of the forward primer (5'-GCCATTCTCACCGGATTCAGTCGTC-3 '). Or modified second primer (SEQ ID NO: 5'-AGCCGCCGTCCCGTCAAGTCAG-3 ') by adding another second zip code sequence (5'-CGACGCGGGCTTGGTACGTTTGGGG-3') to the 5 'end of the reverse primer (SEQ ID NO: 5'-AGCCGCCGTCCCGTCAAGTCAG-3'). 5) is produced (see FIG. 4). In this embodiment, both of the forward modified second primer and the reverse modified second primer were prepared, but only primers in the desired direction of extension can be modified by adding a zip code sequence.

4) That is, as shown in FIG. 1, the second zip code sequence of the forward modified second primer is complementary in a reverse direction to the first zip code sequence of the reverse modified first primer prepared in Example 1-1. (That is, complementary to each other when based on the 5'-3 'direction), and this can be added to the 5' end of the forward primer to produce a forward modified second primer. Further, the second zip code sequence of the reverse modified second primer is complementary to the first zip code sequence of the forward modified first primer prepared in Example 1-1 (that is, in the 5'-3 'direction). Complementary to each other when used as a reference), and this can be added to the 5 'end of the reverse primer to produce a reverse modified second primer.

The sequence of the modified second primer pair produced by modifying the zip code sequence is as follows. The underlined portion of the base sequence of the modified second primer pair below is a zip code sequence.

① modified second primer in the forward direction:

5'- CATGAGCAAGCTGCAGCTGCGCGCG GCCATTCTCACCGGATTCAGTCGTC-3 '(SEQ ID NO: 4)

② modified second primer in reverse direction:

5'- CGACGCGGGCTTGGTACGTTTGGGG AGCCGCCGTCCCGTCAAGTCAG-3 '(SEQ ID NO: 5)

5) To carry out the PCR reaction, 2X PCR Master mix solution (i-StarMAX II) (purchased from Intron Biotechnology Co., Ltd., Seongnam, Gyeonggi-do, Korea) was extended to 12 μl. and GoTaq ^® PCR DNA template) 1㎕ of Core System II, the 3) prepared in process variants a second pair of primers (SEQ ID NO: 4 and SEQ ID NO: 5), insert the 2㎕, total reaction volume by adding distilled water 9㎕ 2 Adjust to 24 μl. And the PCR reaction is carried out under the following conditions, and the temperature is maintained at 4 ℃ after the PCR reaction.

95 ℃ 5 minutes

95 ° C 30 seconds, 55 ° C 30 seconds, 72 ° C 30 seconds (30 cycles)

72 ° C. 5 minutes.

6) In Figure 4 a 5) the process the PCR amplification process, and there is an overview of the amplification product, if hayeoteul amplify the DNA template of GoTaq ^® PCR Core System II by the general forward and reverse primers include poly for extended nucleotide is carried out in While the PCR amplification product has a nucleotide sequence length of 323 bp, when the extension polynucleotide is amplified using the modified second primer pair of the present embodiment, the PCR amplification products of the extension polynucleotide are 5 'end and 3' end. An zip code sequence and a sequence complementary thereto are further formed (323 bp + 25 bp + 25 bp) to have a base sequence length of 373 bp. At this time, the first collection of the amplified target polynucleotide obtained in Example 1-1 at the 5 'end and / or 3' end of the PCR amplification product of the extension polynucleotide amplified using the modified second primer pair of the present embodiment A base sequence capable of binding complementarily to a code sequence or a sequence complementary thereto (in this embodiment, a second zip code sequence or a sequence complementary thereto) is further extended.

7) As described above, the extended polynucleotides amplified and extended may be stored as 373 bp ds-DNA through DNA purification, or may be separated and stored as ss-DNA by performing PCR using magnetic beads or the like according to the purpose. . The ss-DNA separation method using magnetic beads and the like is described in detail in Example 2-2 described later.

Example 1-3 (Step 3): Extension of Base Sequence of Target Polynucleotide Using Amplified Extended Polynucleotide

1) PCR amplification products of the target polynucleotides amplified and extended in step 1 of Example 1-1 can be purified and used for extension reactions using the extended polynucleotides amplified and extended in step 2 of Example 1-2. In addition, the PCR solution of the amplified and extended target polynucleotide can be used for extension reaction.

2) To carry out the PCR reaction, amplified and extended target in step 1 in 25 μl of 2X PCR Master mix solution (i-StarMAX II) (purchased from Intron Biotechnology, Inc., Seongnam, Gyeonggi-do, Korea) 5 μl of the PCR amplification product solution of polynucleotide and 5 μl of the PCR amplification product solution of the extended polynucleotide amplified and extended in step 2 are added, and 15 μl of the second distilled water is added to adjust the total reaction volume to 50 μl. And the PCR reaction is carried out under the following conditions, and the temperature is maintained at 4 ℃ after the PCR reaction.

95 ℃ 5 minutes

95 ° C 30 seconds, 60 ° C 30 seconds, 72 ° C 30 seconds (10 cycles)

95 ° C 30 seconds, 68 ° C 30 seconds, 72 ° C 30 seconds (20 cycles)

72 ° C. 5 minutes.

3) FIG. 5 schematically illustrates the PCR amplification process and the amplification product performed in step 2), wherein the extended poly amplified in step 2 to the PCR amplification product of the target polynucleotide amplified and extended in step 1 PCR reaction under PCR conditions as described above with the addition of ss-DNA or ds-DNA of nucleotides results in 3 complementary to the first zip code sequence of the forward modified first primer in the amplified and extended target polynucleotide. The 'terminal sequence' and the 3 'terminal sequence complementary to the second zip code sequence of the reverse modified second primer in the amplified extended polynucleotide are mutually complementarily complementary and annealed. As described above, in the present invention, the first zip code sequence of the forward modified first primer is complementary to the reverse of the second zip code sequence of the reverse modified second primer (ie, referred to the 5'-3 'direction). Complementary to each other when alternating), the first zip code sequence of the reverse modified first primer is complementary in the opposite direction to the second zip code sequence of the forward modified second primer (i.e., in the 5'-3 'direction). Complementary to each other when used as a reference) because the modified first primer and the modified second primer were designed.

4) Amplified and extended target poly by annealing while the 3 'end of the amplified and extended target polynucleotide and the 3' end of the amplified extended extension polynucleotide are complementary to each other as shown in FIG. The DNA backbone of each of the nucleotides and the extended polynucleotides amplified and extended serves as a primer for PCR amplification with respect to each other, and a double extension reaction is extended in both directions about the base sequences staggered to each other. Dual-Extension) is performed. As a result, a target polynucleotide is produced that is extended and amplified in a nonspecific manner by the extending polynucleotide.

5) On the other hand, by selectively modifying the primers in Steps 1 and 2 according to the purpose, selectively modifying the first primer in the reverse direction and the second modified primer in the reverse direction, or the reverse modified first primer and the forward modified second primer The target polynucleotide can be extended and amplified only in one direction (5 'direction or 3' direction). In addition, the extension direction of the target polynucleotide may be determined depending on whether the amplification product of the extension polynucleotide amplified and extended in step 2 is added in ss-DNA form or ds-DNA form.

6) In addition, it is possible to further extend the target polynucleotide by using an extension polynucleotide having a sequence other than the extended extension polynucleotide amplified in step 2, and using the extended target polynucleotide obtained in step 3 Repeating step 1 to step 3 can further extend the base sequence of the target polynucleotide by a desired length.

7) Meanwhile, Step 1 of Example 1-1, Step 2 of Example 1-2, and Step 3 of Example 1-3 may be performed separately, but may be performed at the same time under the same PCR conditions. It may also be performed by multiplex PCR on target polynucleotides (see Example 4 below). In this case, by modifying the first primer so that the first zip code sequence or the complementary sequence is added to the target polynucleotide to improve detection sensitivity, a distinctive detection signal can be obtained from other target polynucleotides. See Example 4 and FIG. 11).

8) Therefore, according to one embodiment of the present invention, the base sequence of the target polynucleotide may be extended by the required base sequence length, and in particular, the target polynucleotide may be extended nonspecifically regardless of the type of target polynucleotide to be extended. There is an advantage that can be applied to a variety of target polynucleotides present in a variety of samples.

Example 2: Extension of Base Sequence of Target Polynucleotide Immobilized on Solid Phase

As an example, in this embodiment, amplifying a target polynucleotide of interest based on symmetrical or asymmetric solid phase-PCR (SP-PCR), and then extending the amplified target polynucleotide in a nonspecific manner by a desired length. Extend with. Since the target polynucleotide is immobilized on a solid phase surface such as a metal surface, it is similar to the method of Example 1, and thus the detailed description thereof will be omitted.

Example 2-1 (Step 1): Generation of Target Polynucleotide Immobilized on Solid Phase

1) In step 1, a base sequence having no homology with the nucleotide sequence of the genomic DNA at the end of the first primer specifically binding to the target polynucleotide, the first non-homologous nucleotide sequence that does not complementally hybridize with the genomic DNA [ Hereinafter, a modified first primer, which is a primer to which "also referred to as a" zip code sequence "" is added, is prepared.

2) Then, as an example, one of a forward primer and a reverse primer, for example, a PEG (polyethylene glycol) or a carbon chain (-CH _2- ) spacer is bonded to the 5 'end of the forward primer and thiol at the end thereof. After coupling a functional group such as a thiol group, the forward primer is fixed to a solid phase surface such as a metal electrode surface formed in a biosensor chip using a SAM (Self Assembled Method) method using a thiol group. Using a micro-electro-mechanical-systems (MEMS) technique well known in the art, a gold thin film having a size of 700 μm × 700 μm and a thickness of 3,000 μm is prepared, and the 5 ′ end of the forward primer is fixed thereon. . The metal electrode formation and the metal electrode fixing of the nucleotide oligomer can be easily carried out according to a known technique by those skilled in the art, so the detailed description thereof will be omitted (see Korean Patent Nos. 10-0969667 and 10-0969671, etc.).

3) For the preparation of the modified first primer, the base sequences of the forward primer and the reverse primer specific for the 5 'end and the 3' end of the target polynucleotide are determined, respectively. In addition, the first modified first primer (SEQ ID NO: 1) modified by adding one first zip code sequence (5'-CCCCAAACGTACCAAGCCCGCGTCG-3 ') to the 5' end of the forward primer (5'-GCAGCTCCAATGAGAACCTC-3 '). Or modified by changing another primer (SEQ ID NO: 5'-CGCGCGCAGCTGCAGCTTGCTCATG-3 ') by adding another first zip code sequence (5'-CGCGCGCAGCTGCAGCTTGCTCATG-3') to the 5 'end of the reverse primer (5'-AACGAAAAGGTTGGGGTCAT-3'). 2) can be produced.

4) However, in the present embodiment, a primer fixed to a solid surface such as a metal surface, that is, a first primer in a forward direction, may selectively add a zip code sequence such as 5'-CCCCAAACGTACCAAGCCCGCGTCG-3 'to the 5' end. For efficient amplification of the target polynucleotide of interest, it is desirable to amplify the target polynucleotide on a solid phase without modification to the zip code sequence. Accordingly, unlike Example 1-1, the first primer in the forward direction fixed to the solid surface such as the metal surface in step 1 of this Example 2-1 is selected as a zip code modified sequence or an unmodified sequence as follows. It can be designed as.

① forward modified first primer (underlined zip code sequence):

5'-SH-PEG- CCCCAAACGTACCAAGCCCGCGTCG GCAGCTCCAATGAGAACCTC-3 '

or

② forward unmodified first primer:

5'-SH-PEG-GCAGCTCCAATGAGAACCTC-3 '

5) In addition, the reversely modified first primer that is not fixed to a solid phase surface such as a metal surface and is freely present in the buffer for PCR amplification is an oligonucleotide without a spacer and a functional group, and is 5'-CGCGCGCAGCTGCAGCTTGCTCATG- at the 5 'end. It is designed by adding a zip code sequence such as 3 '.

※ Reverse Modified First Primer:

5'- CGCGCGCAGCTGCAGCTTGCTCATG AACGAAAAGGTTGGGGTCAT-3 '

6) On the other hand, when performing an asymmetric SP-PCR reaction, a forward modified first primer or a forward unmodified first primer, which is not fixed to the solid phase surface and is freely present in the buffer for PCR amplification, is required. Unlike the forward modified first primer or the immobilized forward unmodified first primer, the spacer is not attached to the functional group so that it is freely present in the solution, but relatively lower than the reverse modified first primer for the asymmetric PCR reaction. , For example, in a solution in a ratio of 1: 8 or 1:16.

7) In this embodiment, PCR reaction conditions such as PCR reaction composition, reaction time and reaction temperature setting are the same as PCR reaction conditions such as PCR reaction composition, reaction time and reaction temperature setting in Example 1-1. Do this. 6 (a) to 6 (e) schematically illustrate the PCR amplification process and the amplification products performed in this embodiment. According to this embodiment, PCR of a target polynucleotide immobilized on a solid phase surface is performed by performing PCR amplification using a forward first primer or a forward modified first primer immobilized on a solid phase surface and a free reverse modified first primer. At the same time as the amplification product is generated, any desired base sequence (3'-GCGCGCGTCGACGTCGAACGAGTAC-5 ', which is a complementary sequence to the zip code sequence of 5'-CGCGCGCAGCTGCAGCTTGCTCATG-3' in this example) is further extended. Is generated (see FIG. 6E). This annealed with a sequence complementary to the zip code sequence generated by further extension to the extension polynucleotide in the amplification process of the extension polynucleotide to be described later.

Example 2-2 (Step 2): Amplification and Storage of a Single Chain Extended Polynucleotide

1) This example amplifies and stores the extension polynucleotide in a manner similar to step 2 of Example 1-2. That is, the amplification DNA template (323bp) of Promega GoTaq ^® PCR Core System II is prepared by amplifying a polynucleotide for extension, and the polynucleotide for extension can be produced and stored in large quantities. However, unlike step 2 of Example 1-2, it is recommended to store the amplified DNA as ss-DNA. This has the advantage of increasing the yield and shortening the reaction time by extending the target polynucleotide in a non-specific manner using ss-DNA rather than ds-DNA in step 3 to be described later. In step 2, magnetic beads (MB) are used to separate and purify the amplified extension polynucleotide into single chain form. This is described as follows.

2) First, in the same manner as in Example 1-2, the forward modified second primer (SEQ ID NO: 4) and the reverse modified second primer (SEQ ID NO: 5) are prepared, but the reverse modified second primer is 5 After preparing with 5'-SH-PEG- CGACGCGGGCTTGGTACGTTTGGGG AGCCGCCGTCCCGTCAAGTCAG-3 ', it is prepared by binding to a spacer and a thiol group at the 5'- end of the'- CGACGCGGGCTTGGTACGTTTGGGG AGCCGCCGTCCCGTCAAGTCAG-3 ', and then the magnetized to be magnetic. In addition, 5′- CATGAGCAAGCTGCAGCTGCGCGCG GCCATTCTCACCGGATTCAGTCGTC-3 ′, which is a forward modified second primer, is prepared in the same manner as in Example 1-2.

3) In the present embodiment, PCR reaction conditions such as PCR reaction composition, reaction time, and reaction temperature setting are the same as PCR reaction conditions such as PCR reaction composition, reaction time, and reaction temperature setting in Example 1-2. Can be performed. That is, the extended polynucleotide for which the amplification target (GoTaq ^® PCR Core System II the DNA template) (323bp), the reverse fixed to the magnetic bead modification of the second primer, the free forward modification of the second primer 2X PCR master mix solution (I-StarMAX II) (purchased from Intron Biotechnology Co., Ltd., Seongnam-si, Gyeonggi-do, Korea) and then subjected to symmetric PCR or asymmetric PCR under the same PCR conditions as in Example 1-2.

4) After 30 cycles of the PCR reaction process in step 3), the magnetic beads are fixed by pulling down the PCR tube using a magnet, and then the supernatant is carefully removed.

5) Then, after filling the PCR tube with 1X PBS buffer again, the process 4) is performed twice more, and finally, the tertiary distilled water is filled into the PCR tube.

6) After the buffer exchange and the final replacement of the tertiary distilled water three times in total, by removing the magnet from the PCR tube, the PCR amplification product immobilized on the magnetic beads floats freely in the tertiary distilled water in the PCR tube. Will exist. This PCR tube is heated to about 95 ° C., and sonication is carried out for 10 seconds while maintaining the temperature at about 95 ° C.

7) While maintaining the constant temperature of about 95 ℃ by using a magnet to pull the magnetic beads to the bottom of the PCR tube to fix it, carefully remove the supernatant of the PCR tube. The extended polynucleotide thus separated additionally has a 5'-CGCGCGCAGCTGCAGCTTGCTCATG-3 'sequence required for the reaction in step 3 described below as amplified ss-DNA (see FIG. 6 (f)). This 5'-CGCGCGCAGCTGCAGCTTGCTCATG-3 'sequence is complementary in a staggered manner to 5'-CATGAGCAAGCTGCAGCTGCGCGCG-3', which is the zip code sequence of the forward modified second primer.

8) Repeat steps 3) to 7) to produce a large amount of ss-DNA of the extension polynucleotide and perform vacuum drying until there is a minimum amount of water, and then UV-vis spectrometer ( UV-vis Spectrometer) is used to measure ss-DNA concentrations and store for use in step 3.

Example 2-3 (Step 3): Extension of Base Sequence of Target Polynucleotide Immobilized on Solid Phase

1) As described above, after amplifying a target polynucleotide of interest based on symmetrical or asymmetrical Solid Phase-PCR (SP-PCR) in Step 1 of Example 2-1, the amplified target polynucleotide was used as Example 2 Using the extended polynucleotide prepared and stored in Step 2 of -2, the desired length of the base sequence is extended. Since the target polynucleotide is fixed to a solid surface such as a metal surface formed in the biosensor chip, it is similar to the method of Example 1-3, and thus the detailed description thereof will be omitted.

2) In step 1 of Example 2-1, the double chain of the target polynucleotide amplified with SP-PCR and immobilized on a solid phase surface such as a metal surface in a chip is denatured into a single chain. At this time, when the solution in the chip is heated to about 95 ° C. and modified into a single chain, the solution is switched from the state of FIG. 6E to the state of FIG. 6F.

3) The ss-DNA separated from the metal surface by the denaturation process leaving only a single chain target polynucleotide fixed on the metal surface is completely removed by washing with a washing buffer at about 90 ° C. or higher.

4) Then, after draining and removing all the solutions present in the biosensor chip with nitrogen gas, PCR was performed to remove the single strand of extension polynucleotide prepared in advance through Step 2 of Example 2-2 so that the final concentration was 2 μM. After adding to the master mix solution (i-StarMAX II), the mixed solution was injected into the biosensor chip where step 1 of Example 2-1 was completed, followed by step 3 of Examples 1-3. Perform PCR reaction under the same PCR conditions as. A target polynucleotide having the 3'-GCGCGCGTCGACGTCGAACGAGTAC-5 'sequence (sequence complementary to the zip code sequence of 5'-CGCGCGCAGCTGCAGCTTGCTCATG-3') at the 3 'end is added to the 5' of the single chain of the extension polynucleotide by this additional sequence. Based on the '-CGCGCGCAGCTGCAGCTTGCTCATG-3' sequence and the 5 '→ 3' orientation, they are reciprocally complemented and annealed.

5) That is, when the PCR reaction is carried out under the PCR conditions as described above, the 3 'end of the target polynucleotide single chain immobilized on the metal surface as shown in Fig. 6 (e) and (f), amplified and extended By annealing the 3 'ends of a single chain of extended polynucleotides complementarily complementary to each other, the DNA backbone of each of the target polynucleotide and the extending polynucleotide functions as a primer of PCR amplification reaction to each other. Dual-extension is performed extending in both directions about the base sequences staggered to each other. As a result, a target polynucleotide is produced that is extended in a nonspecific manner by the polynucleotide for extension.

6) In addition, as in the case of Example 1, it is possible to further extend the target polynucleotide using an extension polynucleotide having a sequence other than the extension polynucleotide amplified and extended in step 2, and the extension obtained in step 3 By repeating step 1 to step 3 again using the prepared target polynucleotide, the base sequence of the target polynucleotide can be further extended to a desired length.

7) Accordingly, according to one embodiment of the present invention, the base sequence of the extended target polynucleotide fixed to the metal surface in the biosensor chip can be extended by the required base sequence length, and in particular, the type of target polynucleotide to be extended. Regardless of whether the target polynucleotide can be extended non-specifically, there is an advantage that it can be applied to various target polynucleotides present in various samples. In particular, according to one embodiment of the present invention, even when PCR amplification is performed in the biosensor chip, the length of the target amplification product can be sufficiently long to be non-specifically irrespective of the PCR amplification target, thereby increasing the difference in charge before and after amplification. Thereby, electrical detection sensitivity or detection reproducibility can be improved.

Example 3 Method of Enhancing Sensitivity of Target Polynucleotide Using Nanoparticles

As an example, in the present embodiment, a target polynucleotide of interest is secured based on symmetrical or asymmetric solid phase-PCR (SP-PCR), and the polynucleotide for extension is extended to the target polynucleotide through nanoparticles such as gold particles. After linking, the nucleotide sequence is extended in a nonspecific manner by the desired nucleotide length to improve detection sensitivity. Except for linking the extension polynucleotide with the target polynucleotide through a nanoparticle, such as gold particles, it is similar to the method of Example 2, the detailed description thereof will be omitted. Meanwhile, in the present embodiment, an example of a PCR reaction chip in which a target polynucleotide is amplified by a primer immobilized on a metal surface has been described, but the method of the present invention hybridizes the hybridization reaction of the target polynucleotide with a probe immobilized on a metal surface. It is also applicable to the reaction chip.

Example 3-1: Performing Step 1 and Step 2

Obtaining the target polynucleotide immobilized on the surface of the solid phase is carried out substantially the same process as in Example 2-1 (see Fig. 7), and amplification and storage of the single chain extension polynucleotide is also performed in Example 2- The process is carried out substantially the same as 2. Therefore, detailed description of step 1 and step 2 is omitted.

Example 3-2 Attaching Thiol Modified Nucleotide Oligomers to Gold Particles

In this embodiment, the 3 'end of the single chain of the target polynucleotide immobilized on the metal surface and the 3' end of the single chain of the amplified extended polynucleotide are not annealed so as to be complementarily complementary to each other. Through the same nanoparticles, the extension polynucleotide is linked with the target polynucleotide immobilized on the metal surface. Gold particles having this function are prepared by attaching thiol-modified nucleotide oligomers as follows.

1) First, nucleotide oligomers attached to gold particles are modified with thiols. For this purpose 3'-GCGCGCGTCGACGTCGAACGAGTAC-5 '(sequence complementary to the zip code sequence of 5'-CGCGCGCAGCTGCAGCTTGCTCATG-3'), which is an extended sequence further to the 3 'end of the target polynucleotide amplified in step 1 of Example 2-1 The nucleotide oligomeric sequence complementary to the above was determined, and 5'-CGCGCGCAGCTGCAGCTTGCTCATG-3 ', which was an extended sequence at the 3' end of the single chain extension polynucleotide amplified in bulk in Step 2 of Example 2-2 After determining the complementary reverse nucleotide oligomer sequences, each nucleotide oligomer is constructed. A thiol group is then attached to the 5 'end to make each modified nucleotide oligomer. Then, these nucleotide oligomers are reduced with DTT (Dithiothreitol) to convert disulfide bonds existing between these nucleotide oligomers to -SH groups ("-SS-" → "-SH"). A nucleotide oligomer having a functional group attached thereto is obtained. On the other hand, nucleotide oligomers can be modified in addition to thiol groups to functional groups for attaching various nanoparticles, such as functional polymers.

2) 450 μl of commercially available gold particle colloid (0.01% gold chloride concentration, 100 ml volume; purchased from BBInternational, UK) was modified with thiol in step 1) and the final concentration was adjusted to 1 μM. 50 μl of the total volume is mixed with 50 μl of the prepared nucleotide oligomer. Then, the mixed solution is incubated at 25 ° C. for 24 hours and continuously stirred at 100 rpm to immobilize the nucleotide oligomer to the gold particle colloid.

3) Then, 500 μl of Aging Buffer (0.1 M Nacl + 0.01 M sodium phosphate) is added to the mixed solution and incubated at room temperature for 40 hours. It is also washed with 50 mM sodium phosphate and 1 M NaCl in SPSC buffer (pH 6.5) to remove nucleotide oligomers that are non-covalently bound to the gold particles.

4) After centrifuging the mixed solution obtained in step 3) at 13,200rpm for 30 minutes, precipitate gold particles to which nucleotide oligomers are attached, remove the supernatant, and add 500 μl of aging buffer to mix. .

5) The above 4) process is repeated twice to obtain gold particles to which nucleotide oligomers modified with thiol are attached.

6) Take 250 µl of the total volume for the test and measure the absorption wavelength of the gold particles with UV-vis spectrometer. Comparing the absorption wavelength before and after the thiol-modified nucleotide oligomer is attached to the gold particle, the absorption wavelength is shifted toward the longer wavelength by attaching the thiol-modified nucleotide oligomer to the gold particle (see Korean Patent No. 10-0590546). . Therefore, the degree of attachment of the nucleotide oligomer modified with thiol to the gold particles due to the difference in the color of the mixed solution can be seen, and if the adhesion is well, it will appear red. However, the color may change depending on pH or salt. On the other hand, when checking the degree of attachment of nucleotide oligomers modified with thiol to the gold particles by UV-vis spectrometer, the aging buffer is added slowly and mixed slowly to prevent gold particles from agglomerating with each other.

7) The mixed solution of the gold particles to which the nucleotide oligomers modified with thiol obtained through this Example were attached was centrifuged at 13,200 rpm for 30 minutes after replacing the buffer with 1X PBS buffer before use in Step 3 described below. Remove the supernatant, then add 250 μL of 1 × PBS buffer. Repeat this process one more time and add 1X PBS buffer to adjust the total volume to 250 μl.

8) Meanwhile, in the present embodiment, gold particles are used as examples of the particles, but the present invention is not limited thereto. For example, iron oxide nanoparticles, superparamagnetic nanoparticles, silica nanoparticles, magnetic beads, and iron oxides are used. Particles of various sizes and materials known in the art, such as including polymer beads, may be used. That is, any particle that is widely used in the art for therapeutic or research purposes may be used.

Example 3-3 (Step 3): Nonspecific sequencing of target polynucleotide and improvement of detection sensitivity

1) The target polynucleotide obtained on the basis of symmetrical or asymmetrical Solid Phase-PCR (SP-PCR) in Step 1 of Example 3-1 as described above was modified with thiols as prepared in Example 3-2. Nucleotide oligomers are associated with extension polynucleotides prepared in advance via the gold particles to which they are attached (see FIG. 8).

2) For this purpose, the double-chain of the target polynucleotide immobilized on the solid phase surface such as the metal surface in the chip through the SP-PCR process to denature into a single chain. At this time, when the solution in the chip is heated to about 95 ° C. and modified into a single chain, the solution is switched from the state of FIG. 7E to the state of FIG. 8F.

3) And the PCR reaction is carried out in the same manner and in the same manner as in Example 2-3, the gold particles to which the nucleotide oligomers modified with thiol prepared in Example 3-2 are attached are prepared by PCR master mix solution ( After addition to i-StarMAX II), the mixed solution is injected into the biosensor chip in which step 2) is completed, and then PCR reaction is performed under the same PCR conditions as in Step 3 of Example 2-3. At this time, of the nucleotide oligomers attached to the gold particles, the forward nucleotide oligomer is annealed and extended complementarily with the 3'-GCGCGCGTCGACGTCGAACGAGTAC-5 'sequence added to the 3' end of the target polynucleotide (Fig. 8 (f)). And (g)).

4) In addition, after adding a single strand of the extension polynucleotide prepared in advance as in Example 2-2 to the PCR master mix solution (i-StarMAX II), the mixed solution was added to step 3). After the process is injected into the completed biosensor chip, the PCR reaction is performed under the same PCR conditions as in Step 3 of Example 2-3. In this case, the reverse nucleotide oligomers among the nucleotide oligomers attached to the gold particles are annealed and extended by binding complementarily with the 5'-CGCGCGCAGCTGCAGCTTGCTCATG-3 'sequence added to the 3' end of the extending polynucleotide (Fig. 8 (h) ) And FIG. 9).

5) In other words, when the PCR reaction is performed under the PCR conditions as described above, nucleotide oligomers attached to the gold particles at the 3 'end of the target polynucleotide fixed to the metal surface as shown in (f) of FIG. The heavy forward nucleotide oligomer is annealed and extended to primarily bind the target polynucleotide and the gold particles fixed to the metal surface. The reverse nucleotide oligomers of the nucleotide oligomers attached to the gold particles are then annealed and extended at the 3 ′ end of a single chain extension polynucleotide prepared in advance so that the gold particles and the extension polynucleotides are secondarily bound. As a result, the target polynucleotide immobilized on the metal surface has the effect of extending the base sequence in a nonspecific manner by the polynucleotide for extension through the gold particles.

6) In addition, an extended nucleotide sequence annealed to the reverse nucleotide oligomer attached to the gold particles can be further extended by using an extension polynucleotide. In this case, the target polynucleotide may be linked to the polynucleotide for extension through gold particles and may extend the base sequence by a desired base sequence length in a nonspecific manner to improve detection sensitivity. In particular, by adjusting the number of extension polynucleotides associated with the target polynucleotide according to the number of nucleotide oligomers attached to the gold particles, detection sensitivity or detection reproducibility can be improved. In addition, the gold particles may be attached with additional reverse nucleotide oligomers complementary to the extension polynucleotide having a sequence other than the extension polynucleotide previously prepared in step 2.

7) Thus, according to one embodiment of the present invention, the target polynucleotide and the extension polynucleotide can be linked through the nucleotide oligomers attached to the gold particles, thereby allowing the target to be non-specifically regardless of the type of the target polynucleotide. Not only can improve the detection sensitivity by extending the nucleotide sequence of the polynucleotide, but also can adjust the number and length of the extension polynucleotide associated with the target polynucleotide can be applied to a variety of target polynucleotides present in a variety of samples There is this. In particular, according to an embodiment of the present invention, by using a plurality of oligomers attached to the particles, the base sequence can be non-specifically extended, thereby improving detection sensitivity or detection reproducibility, and for nanoparticles or extension such as silica particles. Attaching a labeling material such as a fluorescent material to the polynucleotide can also enhance the detection signal such as an optical signal.

Example 4 Allele and SNP Analysis of the Cytochrome Gene as a Target Polynucleotide

In this example, it is intended to verify that the present invention can be usefully used for allele type analysis and monobasic polymorphism (hereinafter referred to as SNP) analysis of the cytochrome family CYP2C9 and CYP2C19 genes known to be involved in important drug metabolism. .

Example 4-1 Primer Sequencing and Preparation for Target Polynucleotide Amplification

(1) Primer sequencing and preparation for CYP2C9 * 3 allele type amplification

CYP2C9 * 3 is an allele with an A> C SNP at position 1075 based on the cytochrome overall gene map with a base sequence length of 291 bp. Target polynucleotides and primers are prepared using the CYP2C9 * 3 allele type nucleotide sequences well known in the art (see Korean Patent Nos. 10-1024031 and 10-0921793).

The base sequences of the forward and reverse primers specific for the 5 'end and the 3' end of the target polynucleotide are determined as follows.

① CYP2C9 * 3 forward primer:

5'-GAGCCACATGCCCTACAC-3 '(SEQ ID NO: 6)

② CYP2C9 * 3 reverse primer:

5'-ACAAACCTTTATAGCCCCAAAC-3 '(SEQ ID NO: 7)

In addition, as described in Example 1, the first zip code sequence (5'-CGCGCGCAGCTGCAGCTTGCTCATG-3 ') was modified by adding the first zip code sequence to the 5' end of the CYP2C9 * 3 reverse primer (5'-AACGAAAAGGTTGGGGTCAT-3 '). A modified first primer of 5′- CGCGCGCAGCTGCAGCTTGCTCATG ACAAACCTTTATAGCCCCAAAC-3 ′ (SEQ ID NO: 8) was prepared.

(2) Primer sequencing and preparation for CYP2C19 * 2 allele type amplification

CYP2C19 * 2 is an allele with a sequence length of 293 bp (similar in size to CYP2C9 * 3 described above) and an SNP of G> A at position 681, based on the cytochrome total gene map. Target polynucleotides and primers are prepared using the CYP2C19 * 2 allele type nucleotide sequence well known in the art (see Korean Patent Nos. 10-1024031 and 10-0921793).

The base sequences of the forward and reverse primers specific for the 5 'end and the 3' end of the target polynucleotide are determined as follows.

① CYP2C19 * 2 forward primer:

5'-CCAGAGCTTGGCATATTGTATC-3 '(SEQ ID NO: 9)

② CYP2C19 * 2 reverse primer:

5'-AATACGCAAGCAGTCACATAAC-3 '(SEQ ID NO: 10)

In addition, as described in Example 1, the first zip code sequence (5'-CGCGCGCAGCTGCAGCTTGCTCATG-3 ') was modified at the 5' end of the CYP2C19 * 2 reverse primer (5'-AATACGCAAGCAGTCACATAAC-3 '). A modified first primer (5'- CGCGCGCAGCTGCAGCTTGCTCATG AATACGCAAGCAGTCACATAAC-3 ') (SEQ ID NO: 11) was prepared.

(3) Primer sequencing and preparation for CYP2C19 * 3 allele type amplification

CYP2C19 * 3 is an allele with a nucleotide sequence length of 317 bp and a SNP of G> A at position 636 based on the cytochrome overall gene map. Target polynucleotides and primers are prepared using the CYP2C19 * 3 allele type nucleotide sequence well known in the art (see Korean Patent Nos. 10-1024031 and 10-0921793).

The base sequences of the forward and reverse primers specific for the 5 'end and the 3' end of the target polynucleotide are determined as follows.

① CYP2C19 * 3 forward primer:

5'-CACCCTGTGATCCCACTTTC-3 '(SEQ ID NO: 12)

② CYP2C19 * 3 reverse primer:

5'-AGAACTTTGCCATCTTTTCCAG-3 '(SEQ ID NO: 13)

In addition, as described in Example 1, the first zip code sequence (5'-CGCGCGCAGCTGCAGCTTGCTCATG-3 ') was modified at the 5' end of the CYP2C19 * 3 reverse primer (5'-AGAACTTTGCCATCTTTTCCAG-3 '). A modified first primer of 5′- CGCGCGCAGCTGCAGCTTGCTCATG AGAACTTTGCCATCTTTTCCAG-3 ′ (SEQ ID NO: 14) was prepared.

Meanwhile, in Example 4-1, both a forward modified first primer and a reverse modified first primer may be prepared, but for the purpose of performing multiplex PCR on a plurality of target polynucleotides, a selective extension is desired. Only primers in the direction, ie reverse, were modified by adding the first zip code sequence. This reverse modified first primer corresponds to the forward modified second primer of the polynucleotide for extension described later, the first zip code sequence of the reverse modified first primer is the second set of the forward modified second primer described later It is designed to be complementary in reverse to the code sequence (ie staggered complementary to each other based on the 5′-3 ′ direction) (see FIG. 10).

Example 4-2 Primer Sequencing and Preparation for Extension Polynucleotide Amplification

In this embodiment, using the DNA template (323bp) of ^{GoTaq ® PCR Core System II (Promega} , M7665) Promega Corporation as a polynucleotide for extension. Amplification and storage of such extension polynucleotides are described in detail in Examples 1-2 and 2-2, and thus description thereof is omitted.

The base sequences of the forward and reverse primers specific for the 5 'end and the 3' end of the extension polynucleotide are determined as follows.

① extension polynucleotide (ES) forward primer:

5'-GCCATTCTCACCGGATTCAGTCGTC-3 '(SEQ ID NO: 15)

② extension polynucleotide (ES) reverse primer:

5'-AGCCGCCGTCCCGTCAAGTCAG-3 '(SEQ ID NO: 16)

In addition, as described in Example 2, a second zip code sequence (5'-CATGAGCAAGCTGCAGCTGCGCGCG-3 ') is provided at the 5' end of the forward primer (5'-GCCATTCTCACCGGATTCAGTCGTC-3 ') of the extension polynucleotide (ES). In addition, a modified modified second primer (5′- CATGAGCAAGCTGCAGCTGCGCGCG GCCATTCTCACCGGATTCAGTCGTC-3 ′) (SEQ ID NO: 4) was prepared.

Meanwhile, in Example 4-2, both a forward modified second primer and a reverse modified second primer may be prepared, but for the purpose of performing multiplex PCR on a plurality of target polynucleotides, an optional extension is desired. Only the primer in the direction, ie forward, was modified by adding the second zip code sequence. This forward modified second primer corresponds to the reverse modified first primer of the target polynucleotide described above, and the second zip code sequence of the forward modified second primer is the first zip code of the reverse modified first primer described above. Complementary to the sequence in reverse (ie staggered complementary to each other based on the 5′-3 ′ direction) (see FIG. 10).

Example 4-3 Amplification of Cytochrome CYP2C9 * 3, CYP2C19 * 2 and CYP2C19 * 3 Alleles Using Conventional Single PCR and Multiplex PCR Methods

1) In the present embodiment, the CYP2C9 * 3 genomic gene, CYP2C19 * 2 genomic gene, CYP2C19 * 3 genomic gene, extension polynucleotide (ES) prepared in Examples 4-1 and 4-2, and amplification thereof, respectively. Single and multiplex PCR were performed using the forward and reverse primers.

2) To this end, the CYP2C9 * 3, CYP2C19 * 2 and / or CYP2C19 * 3 genome genes (2X PCR Master mix solution (i-StarMAX II) (purchased from Intron Biotechnology, Inc., Seongnam, Gyeonggi-do, Korea) gDNA), the extension polynucleotide (ES) and each of the forward and reverse primers for amplifying them are added in a single PCR composition ratio and multiplex PCR composition ratio as shown in Table 1 below to adjust the total reaction volume to 20 μl. And the PCR reaction is carried out under the following conditions, and the temperature is maintained at 4 ℃ after the PCR reaction.

95 ℃ 5 minutes

95 ° C 30 seconds, 57 ° C 90 seconds, 72 ° C 60 seconds (35 cycles)

72 ° C. 7 minutes.

Table 1

3) After performing a single PCR and multiplex PCR of step 2), gel electrophoresis (1.5% agarose gel, 2, 5 and 10 μl of sample and 3 μl of marker (100bp) loading) was performed. . The result is as shown in the electrophoresis picture on the left of FIG. Bands of these gene amplification products in gel electrophoresis because the size of the cytochrome CYP2C9 * 3 gene (291 bp) and the size of the CYP2C19 * 2 gene (293 bp) are almost the same when the conventional multiplex PCR is used as in this example. Could not be distinguished by overlapping.

Example 4-4: Band discrimination of cytochrome CYP2C9 * 3, CYP2C19 * 2 and CYP2C19 * 3 allele-type amplification and amplification products by multiplex PCR method according to the present invention

1) First, in this embodiment, the CYP2C9 * 3 genomic gene, CYP2C19 * 3 genomic gene, extension polynucleotide (ES) prepared in Examples 4-1 and 4-2, and respective forward primers for amplifying them, and Single PCR was performed under the same conditions as in Example 4-3 using the reverse primer. Single PCR composition ratio is shown in Table 2 below. After performing a single PCR of the procedure, gel electrophoresis (1.5% agarose gel, 2, 5 and 10 μl of sample and 3 μl of marker (100bp) loading) was performed. The result is as shown in the electrophoresis picture of the right side of FIG.

TABLE 2

2) CYP2C9 * 3, CYP2C19 * 2 and / or CYP2C19 * 3 genome genes (2X PCR Master mix solution (i-StarMAX II) (purchased from Intron Biotechnology, Inc., Seongnam, Gyeonggi-do, Korea) gDNA), extended polynucleotides (ES), and forward and reverse primers for amplifying them were added to adjust the total reaction volume to 20 µL, and then multiplex PCR was performed under the same conditions as in Example 4-3. . However, the difference from Example 4-3 is a modified primer modified by adding a zip code sequence as a reverse primer of the CYP2C9 * 3 genomic gene and a forward primer of an extension polynucleotide (ES) as shown in Table 3 below. (SEQ ID NO: 8 and SEQ ID NO: 4, respectively).

TABLE 3

3) That is, in the multiplex PCR of 2), the cytochrome CYP2C9 * 3 genomic gene was used as the reverse primer, and the reverse modified first primer of SEQ ID NO: 8, and the extension polynucleotide (ES) was sequenced as the forward primer. A forward modified second primer of number 4 was used. Therefore, in the multiplex PCR of step 2), the cytochrome CYP2C9 * 3 genomic gene is extended and amplified by a dual extension method as illustrated in FIG. 10. That is, when amplifying the cytochrome CYP2C9 * 3 genomic gene in a conventional manner, an amplification product of 316 bp is generated, but when it is amplified by the dual extension method of FIG. 10, a 639 bp amplification product is generated.

4) gel electrophoresis (1.5% agarose gel, 2, 5 and 10 μl sample and 3 μl marker) after performing multiplex PCR of step 2) to confirm the amplification result of step 3). (100 bp) loading). The result is as shown in the electrophoresis picture of the right side of FIG. Using conventional multiplex PCR, the cytochrome CYP2C9 * 3 gene size (291bp) and the CYP2C19 * 2 gene size (293bp) are almost the same, so that the bands of these gene amplification products can be distinguished in gel electrophoresis. On the other hand, when amplified by multiplex PCR using the dual extension of FIG. 10, the CYP2C9 * 3 gene band was removed from the CYP2C19 * 2 gene band because the cytochrome CYP2C9 * 3 gene was nonspecifically extended. It can be seen that it can be easily separated and analyzed (right).

5) Therefore, according to one embodiment of the present invention, even when performing multiplex PCR on a plurality of gene templates, non-specific extension of a nucleotide sequence to a target polynucleotide to be detected is possible, and accordingly, Improved detection sensitivity can be obtained, and multiplex PCR using this dual extension can be very useful for genotyping and SNP analysis of specific genes having multiple alleles.

Although the present invention has been described with reference to the above embodiments, the present invention is not limited thereto. Those skilled in the art can make modifications and changes without departing from the spirit and scope of the present invention, and it will be appreciated that such modifications and changes also belong to the present invention.

Claims (27)

In the method for non-specifically extending the nucleotide sequence of the target polynucleotide, (a) a first non-homologous sequence not complementarily hybridized with the genomic gene as a base sequence having no homology with the nucleotide sequence of the genomic gene at the end of the first primer specifically binding to the target polynucleotide, or complementary thereto Preparing a modified first primer added with a specific sequence, (b) preparing an extension polynucleotide that is not complementarily hybridized with the target polynucleotide and is used to nonspecifically extend the target polynucleotide; (c) a nucleotide sequence having no homology with the nucleotide sequence of the genomic gene at the end of the second primer that specifically binds to the extension polynucleotide, and is not complementarily hybridized with the genomic gene, but not the hybridization of the modified first primer. 1 preparing a second non-homologous sequence complementary to the non-homologous sequence or a modified second primer added with a sequence complementary thereto; (d) using the modified first primer to add the first non-homologous or complementary sequence to the end of the target polynucleotide to obtain an extended target polynucleotide, and using the modified second primer. Adding a second non-homologous sequence or a base sequence complementary thereto to obtain an extension polynucleotide for extension; (e) the first non-homologous sequence added to the end of the target polynucleotide or the base complementary thereto, and the second non-homologous sequence added to the extension polynucleotide end or the sequence complementary thereto complement A method of non-specifically extending the nucleotide sequence of the target polynucleotide comprising the step of annealing to an enemy to extend the target polynucleotide and the extension polynucleotide obtained in step (d) with respect to each other. In the method for non-specifically extending the nucleotide sequence of the target polynucleotide, (a) a first non-homologous sequence not complementarily hybridized with the genomic gene as a base sequence having no homology with the nucleotide sequence of the genomic gene at the end of the first primer specifically binding to the target polynucleotide, or complementary thereto Preparing a modified first primer added with a specific sequence, (b) preparing an extension polynucleotide that is not complementarily hybridized with the target polynucleotide and is used to nonspecifically extend the target polynucleotide; (c) a nucleotide sequence having no homology with the nucleotide sequence of the genomic gene at the end of the second primer that specifically binds to the extension polynucleotide, and is not complementarily hybridized with the genomic gene, but not the hybridization of the modified first primer. 1 preparing a second non-homologous sequence complementary to the non-homologous sequence or a modified second primer added with a sequence complementary thereto; (d) using the modified first primer to add the first non-homologous or complementary sequence to the end of the target polynucleotide to obtain an extended target polynucleotide, and using the modified second primer. Adding a second non-homologous sequence or a base sequence complementary thereto to obtain an extension polynucleotide for extension; (e) modifying a forward nucleotide oligomer complementary to the first non-homologous sequence or complementary to the target polynucleotide terminus with a functional group for particle attachment, and adding the agent added to the extension polynucleotide terminus. (2) modifying a reverse nucleotide oligomer complementary to a nonhomologous sequence or a sequence complementary thereto to a functional group for particle attachment, (f) attaching the modified forward and reverse nucleotide oligomers to the particles, (g) the forward nucleotide oligomer attached to the particle obtained in step (f) is extended by annealing complementarily with a first non-homologous sequence added to the target polynucleotide termini or a complementary nucleotide sequence, and (f) The reverse nucleotide oligomer attached to the particle obtained in step) is complementarily annealed and extended with a second non-homologous sequence added to the extension polynucleotide end or a sequence complementary thereto. A method of nonspecifically extending a sequence. The method according to claim 1 or 2, The target polynucleotide obtained in the step (d) is a method of non-specifically extending the base sequence of the target polynucleotide, characterized in that the immobilized on the surface of the solid phase. The method according to claim 1 or 2, The modified first primer may be modified by adding one first non-homologous base sequence at the 5 'end of the forward primer, or another first non-homologous base sequence at the 5' end of the reverse primer. A method of non-specifically extending the base sequence of the target polynucleotide, further comprising using a modified modified first primer of the reverse direction, or both. The method of claim 4, wherein The modified second primer is a reverse modified second primer modified by adding a second non-homologous base sequence complementary to the first non-homologous base sequence of the forward modified first primer at the reverse primer 5 'end, Or a forward modified second primer modified by adding a second non-homologous sequence complementary to the first non-homologous sequence of the reverse-modified first primer at the forward primer 5 'end, or both. Method for non-specifically extending the base sequence of the target polynucleotide, characterized in that. The method of claim 5, Depending on the combination of the forward modified first primer and the reverse modified second primer or the combination of the reverse modified first primer and the forward modified second primer, the target polynucleotide is non-specific in one direction only. Non-specifically extending the base sequence of the target polynucleotide, characterized in that extending to. The method according to claim 1 or 2, The extension polynucleotide extended in the step (d) is used in the form of a double chain, or a specific sequence of the target polynucleotide, characterized in that separated by a single chain by using a PCR amplification reaction using magnetic beads How to extend. The method of claim 7, wherein Non-specifically extending the base sequence of the target polynucleotide, characterized in that the extension direction of the target polynucleotide is determined according to whether to use the extended polynucleotide extended in step (d) in the form of a double chain or a single chain How to. The method according to claim 1 or 2, After further amplifying and preparing an extension polynucleotide having another sequence in addition to the extension polynucleotide obtained by extension in step (d), the target polynucleotide obtained in step (d) is further extended. A method of nonspecifically extending the nucleotide sequence of the target polynucleotide. The method of claim 1, And further extending the target polynucleotide extended in step (e) using the extension polynucleotide. The method of claim 2, The reverse polynucleotide oligomer attached to the particle in the step (g) is annealed complementarily with the second non-homologous sequence added to the extension polynucleotide terminal or the sequence complementary thereto, and the extended poly sequence is returned again. A method of non-specifically extending the base sequence of the target polynucleotide further comprising the step of further extending using a nucleotide. The method according to claim 2 or 11, wherein A method of non-specifically extending the base sequence of the target polynucleotide further comprises the step of labeling the labeling material for signal enhancement to the particles or the extension polynucleotide. The method according to claim 2 or 11, wherein A plurality of forward nucleotide oligomers and reverse nucleotide oligomers are attached to the particles, and the target polynucleotide is linked to the target polynucleotide according to the number of nucleotide oligomers attached to the particles. Nonspecifically extending the nucleotide sequence of the polynucleotide. A first non-homologous sequence not complementarily hybridized with the genomic gene as a base sequence having no homology with the nucleotide sequence of the genomic gene at the end of the first primer specifically binding to the target polynucleotide, or a sequence complementary thereto And a modified first primer added with, A base sequence that does not have homology with the nucleotide sequence of the genomic gene at the end of the second primer that specifically binds to the extending polynucleotide used to nonspecifically extend the base sequence of the target polynucleotide, complementarily with the genomic gene. Nonspecific hybridization of the target polynucleotide, including a non-hybridized second non-homologous sequence complementary to the first non-homologous sequence of the modified first primer or a modified second primer added complementary thereto A primer composition for extending to. The method of claim 14, The modified first primer is a forward modified first primer modified by adding one first non-homologous base sequence at the 5 'end of the forward primer, or another first non-homologous base sequence at the 5' end of the reverse primer. A primer composition for non-specifically extending the base sequence of a target polynucleotide comprising a modified first primer in reverse direction or both. The method of claim 15, The modified second primer is a reverse modified second primer modified by adding a second non-homologous sequence complementary to the first non-homologous base sequence of the forward modified first primer at the 5 'end of the reverse primer, or A forward modified second primer modified by adding a second non-homologous sequence complementary to the first non-homologous sequence of the reverse-modified first primer at the forward primer 5 'end, or both Primer composition for nonspecifically extending the nucleotide sequence of the polynucleotide. The method of claim 16, The base sequence of the target polynucleotide is used as a combination of the forward modified first primer and the reverse modified second primer, or a combination of the reverse modified first primer and the forward modified second primer. Primer composition for nonspecific extension. DNA polymerase, dNTPs and buffers, A first non-homologous sequence not complementarily hybridized with the genomic gene as a base sequence having no homology with the nucleotide sequence of the genomic gene at the end of the first primer specifically binding to the target polynucleotide, or a sequence complementary thereto And a modified first primer added with, A base sequence that does not have homology with the nucleotide sequence of the genomic gene at the end of the second primer that specifically binds to the extending polynucleotide used to nonspecifically extend the base sequence of the target polynucleotide, complementarily with the genomic gene. A modified second primer which does not hybridize and adds a second non-homologous sequence complementary to the first non-homologous sequence of the modified first primer or a sequence complementary thereto; An extension polynucleotide that is not complementarily hybridized with the target polynucleotide and is used to nonspecifically extend the target polynucleotide, wherein the second non-homologous sequence or the complementary sequence is added by the modified second primer. Target polynucleotide detection sensitivity kit comprising a polynucleotide for extension. The method of claim 18, The extended polynucleotide is used in the form of a double chain, or a target polynucleotide detection sensitivity improvement kit, characterized in that the separation is used in a single chain by performing a PCR amplification reaction using magnetic beads. The method of claim 18 or 19, The modified first primer is a forward modified first primer modified by adding one first non-homologous base sequence at the 5 'end of the forward primer, or another first non-homologous base sequence at the 5' end of the reverse primer. Kit for improving the detection sensitivity of the target polynucleotide comprising a modified first primer of the reverse direction, or both. The method of claim 20, The modified second primer is a reverse modified second primer modified by adding a second non-homologous sequence complementary to the first non-homologous base sequence of the forward modified first primer at the 5 'end of the reverse primer, or A forward modified second primer modified by adding a second non-homologous sequence complementary to the first non-homologous sequence of the reverse-modified first primer at the forward primer 5 'end, or both Polynucleotide detection sensitivity improvement kit. The method of claim 21, For improving the sensitivity of the target polynucleotide detection, characterized in that the combination of the forward modified first primer and the reverse modified second primer, or a combination of the reverse modified first primer and the forward modified second primer Kit. The method of claim 18 or 19, A target polynucleotide further comprising a particle having a forward nucleotide oligomer complementary to the first non-homologous sequence or a complementary nucleotide sequence and a reverse nucleotide oligomer complementary to the second non-homologous sequence or a sequence complementary thereto Detection sensitivity improvement kit. The method of claim 23, wherein And a further nucleotide oligomer complementary to the extension polynucleotide having a sequence other than the extension polynucleotide to the particle may be attached to the particle. The method of claim 23, wherein The particle or the extension polynucleotide is a target polynucleotide detection sensitivity kit, characterized in that the labeling material for enhancing the signal. The method of claim 1, When performing multiplex PCR on a plurality of gene templates, the base sequence of the target polynucleotide is obtained by non-specifically extending the base sequence of the target polynucleotide to be detected to obtain an improved detection sensitivity for the target polynucleotide. To nonspecifically. The method of claim 1 or 26, A method of non-specifically extending the base sequence of a target polynucleotide, characterized in that it is used for genotyping or SNP analysis of a specific gene having a plurality of allele types.

Images