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KR-20260065025-A - Switchable on-off primers and nucleic acid amplification method using the same

KR20260065025AKR 20260065025 AKR20260065025 AKR 20260065025AKR-20260065025-A

Abstract

The present invention relates to a switchable on-off primer and a nucleic acid amplification method using the same. More specifically, the present invention can be utilized for isothermal amplification of nucleic acids without temperature change by using an on-off primer that can be switched depending on the presence or absence of an analyte.

Inventors

  • 최석정

Assignees

  • 국립강릉원주대학교산학협력단

Dates

Publication Date
20260508
Application Date
20241031

Claims (18)

  1. It includes an analyte binding sequence located at the 5' position that can bind to the analyte and a primer sequence located at the 3' position, The above analyte binding sequence comprises a protruding sequence and a stem sequence sequentially starting from the 5'-terminus, and The entire primer sequence or a portion of its 3'-terminus binds complementarily to the stem sequence to form a double-stranded stem, and One or more selected from the group consisting of a 3'-terminal portion of the analyte binding sequence, a 5'-terminal portion of the primer sequence, and an additional sequence for loop formation, form a loop, and The protruding sequence of the analyte binding sequence is a single-stranded hairpin-structured on-off primer.
  2. In paragraph 1, The analyte binding sequence is an on-off primer selected from a base sequence complementary to the analyte or an aptamer base sequence capable of specifically binding to the analyte.
  3. In paragraph 1, An additional sequence for loop formation is an on-off primer located between the analyte binding sequence and the primer sequence.
  4. In paragraph 1, The on-off primer further includes one or more of a functional sequence and a primer binding sequence, wherein The above functional sequence is a sequence capable of causing an amplification reaction in a double-stranded state, and The above primer binding sequence binds to the primer sequence of the on-off primer and is an on-off primer located at the 3'-terminus of the above functional sequence.
  5. In paragraph 4, An on-off primer, the functional sequence of which is selected from a nicking enzyme recognition sequence, a promoter sequence capable of initiating RNA synthesis, or a sequence capable of generating double-stranded DNA that can act as a substrate for a CRISPR-Cas enzyme.
  6. In paragraph 4, The on-off primer is selected from the sequence of analyte binding sequence-functional sequence-primer binding sequence-primer sequence starting from the 5'-end, the sequence of functional sequence-analyte binding sequence-primer binding sequence-primer sequence, or the sequence of functional sequence-primer binding sequence-analyte binding sequence-primer sequence.
  7. In paragraph 1, the on-off primer is When the analyte binds to the analyte binding sequence, the primer sequence dissociates from the analyte binding sequence and becomes an activated ON state, and An on-off primer, which is a switchable isothermal amplification primer in which the 3'-terminus of the primer sequence is extended by DNA polymerase when the analyte is not bound, resulting in a deactivated off state.
  8. In Paragraph 7, Isothermal amplification can be performed using Nucleic Acid Sequence-based Amplification (NASBA), Strand Displacement Amplification (SDA), Rolling Circle Amplification (RCA), Nicking Enzyme Amplification Reaction (NEAR), exponential amplification reaction (EXPAR), self-priming hairpin-utilized isothermal amplification (SPHIA), or self-priming phosphorothioated hairpin-mediated isothermal. On-off primer, selected from amplification (SP-HAMP).
  9. In paragraph 1, The analyte is an on-off primer that is any one of nucleic acids, proteins, carbohydrates, lipids, organic molecules, metal ions, bacteria, virus particles, or eukaryotic cells.
  10. An on-off primer of any one of claims 1 to 9; DNA polymerase; and A composition for nucleic acid amplification containing dNTPs.
  11. In Paragraph 10, The composition further comprises a template nucleic acid participating in the amplification reaction, wherein the template nucleic acid A primer binding sequence capable of binding to the primer sequence of an on-off primer; and A composition for nucleic acid amplification comprising a functional sequence.
  12. In Paragraph 11, A nucleic acid amplification composition, wherein the functional sequence is selected from a nicking enzyme recognition sequence, a promoter sequence capable of initiating RNA synthesis, or a sequence capable of generating double-stranded DNA that can act as a substrate for a CRISPR-Cas enzyme.
  13. In Paragraph 10, A composition for nucleic acid amplification, comprising one or more of a cutting enzyme and an RNA polymerase.
  14. A nucleic acid amplification method comprising the step of reacting a sample containing an analyte with a nucleic acid amplification composition of any one of claims 10 to 13.
  15. A first step of reacting a sample containing an analyte with a primer solution comprising an on-off primer according to any one of claims 1 to 9; and A nucleic acid amplification method comprising a second step of reacting an amplification solution containing at least one of a cutting enzyme and an RNA polymerase, a DNA polymerase, a template nucleic acid, and a dNTP with the reaction mixture of the first step.
  16. A first step of reacting a sample containing an analyte with a primer solution comprising an on-off primer according to any one of claims 1 to 9; A second step of reacting the reaction mixture of the first step, DNA polymerase, and dNTPs; and A nucleic acid amplification method comprising a third step of reacting at least one of a template nucleic acid, a cutting enzyme, and an RNA polymerase with the reaction mixture of the second step.
  17. In paragraph 15 or 16, A nucleic acid amplification method in which on-off primers are set to a concentration of 1 to 100 times the highest concentration within a certain range of target detection ranges (dynamic range) for the analyte.
  18. In paragraph 15 or 16, A nucleic acid amplification method in which a sample containing an analyte is prepared such that a first step in the sample preparation process is performed by using a primer solution during sample preparation.

Description

Switchable on-off primers and nucleic acid amplification method using the same The present invention relates to an on-off primer capable of switching depending on the presence or absence of an analyte, and a nucleic acid amplification method using the same. In molecular diagnostics for detecting nucleic acids with specific base sequences, the method of amplifying nucleic acids via polymerase chain reaction is widely used. However, this method has the disadvantage of being costly and difficult to apply in the field because it requires equipment for temperature conversion. To address these problems, isothermal nucleic acid amplification methods such as nucleic acid sequence-based amplification (NASBA), strand displacement amplification (SDA), loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HDA), recombinase-primed amplification (RPA), and rolling circle amplification (RCA) have been developed. However, these isothermal amplification methods generally have low robustness and, depending on the method, have disadvantages such as low amplification efficiency, low accuracy, complex components, or limited application range. In particular, among the most commonly used methods, LAMP has the disadvantage of requiring a high temperature of 65°C and making primer design difficult, while RPA has the disadvantage of being sensitive to reaction conditions. FIG. 1 illustrates the process of an on-off primer according to one embodiment of the present invention being activated to become an on primer and being deactivated to become an off primer. FIG. 2 illustrates an amplification process using a template nucleic acid comprising an on-off primer and a functional sequence according to one embodiment of the present invention. FIG. 3 illustrates the process in which a first amplification reaction occurs on the on-off primer itself containing a functional sequence according to one embodiment of the present invention, and the process in which a second amplification reaction occurs when the first amplification product acts on a template containing a functional sequence. Figure 4 illustrates a process in which an on-off primer containing a functional sequence and a template containing a functional sequence directly interact to cause an amplification reaction according to one embodiment of the present invention. FIG. 5 shows one embodiment of the analysis method of the present invention using a template comprising a cutting enzyme recognition sequence as a functional sequence. FIG. 6 shows one embodiment of the analysis method of the present invention using a template comprising a promoter base sequence on the coding strand side as a functional sequence. FIG. 7 illustrates an embodiment of the analysis method of the present invention using a template comprising a promoter base sequence on the template strand side as a functional sequence. FIG. 8 illustrates an embodiment of the analysis method of the present invention using two templates, namely a template containing a cutting enzyme recognition sequence and a template containing a promoter base sequence on the template strand. FIG. 9 illustrates an embodiment of the analysis method of the present invention using an on-off primer containing a functional sequence, an amplification template not containing a functional sequence, and an aptamer template containing a functional sequence. FIG. 10 compares a two-step method and a three-step analysis method in one embodiment of the present invention. Figure 11 analyzes the effect of on-off primer concentration on blank values in one embodiment of the present invention. FIG. 12 shows the results of detecting a target nucleic acid at a concentration of 100 aM when the on-off primer concentration is 20 nM in one embodiment of the present invention. FIG. 13 shows the results of detecting a target nucleic acid at a concentration of 100 aM when the on-off primer concentration is 1 nM in one embodiment of the present invention. Figure 14 shows the result of detecting a target nucleic acid at a concentration of 100 aM when the on-off primer concentration is 1 pM in one embodiment of the present invention. Preferred embodiments of the present invention will be described in detail below with reference to the attached drawings. Prior to this, terms and words used in this specification and claims should not be interpreted as being limited to their ordinary or dictionary meanings, and should be interpreted in a meaning and concept consistent with the technical spirit of the present invention, based on the principle that the inventor can appropriately define the concept of the terms to best describe his invention. Therefore, it should be understood that the embodiments described in this specification and the configurations illustrated in the drawings are merely the most preferred embodiments of the present invention and do not represent all of the technical ideas of the present invention, and that various equivalents and m