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WO2025079958A1 - Procédé de ligature d'adaptateur spécifique d'un mutant à haut rendement - Google Patents

Procédé de ligature d'adaptateur spécifique d'un mutant à haut rendement Download PDF

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Publication number
WO2025079958A1
WO2025079958A1 PCT/KR2024/015269 KR2024015269W WO2025079958A1 WO 2025079958 A1 WO2025079958 A1 WO 2025079958A1 KR 2024015269 W KR2024015269 W KR 2024015269W WO 2025079958 A1 WO2025079958 A1 WO 2025079958A1
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Prior art keywords
probe
base
target dna
mutant
dna
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English (en)
Korean (ko)
Inventor
정철희
김한아
이희경
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Korea University Research and Business Foundation
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Korea University Research and Business Foundation
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Priority claimed from KR1020240134708A external-priority patent/KR20250052964A/ko
Application filed by Korea University Research and Business Foundation filed Critical Korea University Research and Business Foundation
Publication of WO2025079958A1 publication Critical patent/WO2025079958A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6853—Nucleic acid amplification reactions using modified primers or templates
    • C12Q1/6855—Ligating adaptors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes

Definitions

  • the present invention relates to a high-throughput mutation-specific adapter ligation method, and a mutation enrichment method for detecting mutations with low gene frequency, which utilizes two kinds of enzymes, flap endonuclease (hereinafter referred to as FEN1) and HIFI ligase, and enables more sensitive detection of low-frequency mutations at the single nucleotide level by using a probe containing artificial mismatches in addition to the dual enzymatic action, and relates to a novel mutation enrichment technique that secures sufficient reaction efficiency through temperature cycling.
  • FEN1 flap endonuclease
  • HIFI ligase HIFI ligase
  • Such cfDNA is quite low, with an amount of less than 10 ng per ml or 3,000 genome copies that can be obtained from a patient's plasma sample, and has a variant allele frequency (VAF) of 0.01 to 5%, so the analysis method is limited.
  • Representative analysis methods include next generation sequencing (NGS) and droplet digital PCR (ddPCR).
  • NGS is a technology that can read the base sequence quickly and inexpensively by analyzing each nucleic acid strand. NGS has high sensitivity and can detect samples with low VAF, but it requires high sequencing depth, which leads to increased costs.
  • ddPCR is a system that splits the PCR reaction into microdroplets, amplifies them, and counts the signal of each droplet. ddPCR can also detect low VAF in principle, but most commercial systems have a limited detection limit and multiplexing is impossible.
  • nucleic acid analysis technologies such as quantitative real-time PCR, Sanger sequencing, and high resolution melting analysis, but it is impossible to distinguish errors resulting from false positives from actual signals in samples with low VAF.
  • the inventors of the present invention have developed a new technology that can solve the disadvantages of the existing NGS, such as the cost burden resulting from the high sequencing depth, and other disadvantages of nucleic acid analysis technology, such as low sensitivity, while simultaneously detecting various mutation positions, and have devised a new method named HTML (High Throughput Mutant-specific Ligation).
  • HTML High Throughput Mutant-specific Ligation
  • the new method comprises a probe containing an artificial mismatch, which is an essential element of HTML, forming a structure with a DNA template, and FEN1 and HIFI ligase acting sequentially to specifically link a 5' adapter to the base of the mutation position, thereby confirming that the ligase generated according to the base of each mutation position is amplified through a PCR process using a universal primer, and ultimately selectively enriching only the mutation from a mixture of wild type and mutant. Therefore, the present invention has been completed by confirming that the method of the present invention can distinguish the target amplicon with specificity at the single nucleotide level.
  • Another object of the present invention is to provide a method for enriching mutant target DNA, which comprises the steps of hybridizing the probe and universal probe of the present invention with target DNA, treating with FEN1 (flap endonuclease 1) and HIFI ligase to perform adapter ligation, and then performing a PCR amplification reaction using a universal primer.
  • FEN1 overlap endonuclease 1
  • HIFI ligase HIFI ligase
  • the probe may include a base sequence region complementary to a target DNA for binding to the target DNA; and a base sequence region complementary to a universal probe base sequence for binding to the universal probe.
  • the probe can have improved structural identification ability of FEN1 (flap endonuclease 1) by introducing an artificial mismatch base.
  • the target DNA having a mutant base may be cfDNA (cell free DNA).
  • the present invention also provides a composition for capturing mutant target DNA for NGS (Next Generation Sequencing) analysis, comprising the probe of the present invention.
  • NGS Next Generation Sequencing
  • the method may be such that a 5’ adapter is specifically linked to a base at a mutation position.
  • the present invention provides a method for enriching mutant target DNA, including the step of hybridizing the probe and universal probe of the present invention with target DNA, treating with FEN1 (flap endonuclease 1) and HIFI ligase to perform adapter ligation, and then performing a PCR amplification reaction using a universal primer.
  • FEN1 overlap endonuclease 1
  • HIFI ligase HIFI ligase
  • the present invention is a technology that can improve the difficulty of analyzing samples having low sensitivity or low VAF due to high cost, which are problems of existing nucleic acid analysis technologies, by using FEN1 and HIFI ligase and two types of probes to specifically ligate an adapter only to a mutant base (molecule), and successfully enriching mutations through a PCR process using a universal primer after the adapter ligation, thereby easily detecting a mutation to be detected.
  • the technology of the present invention can be applied to various mutation positions simultaneously, and can resolve the complexity of probe design with a simple probe design, and further, the present invention can be usefully utilized in a diagnosis of diseases such as cancer based on liquid biopsy and a mutation detection method that reduces the risk of false positives.
  • Figure 2 illustrates a process of analyzing cfDNA by applying the mutation enrichment method of the present invention.
  • FIG. 4 shows the results of confirming whether efficiency increases through temperature cycling when using a type of U probe without mismatch and a U probe having an N sequence in the 3' sequence in one embodiment of the present invention.
  • Figure 5 shows the results of comparative analysis of the efficacy of adapter ligation at three different reaction temperatures in one embodiment of the present invention.
  • FIG. 6 illustrates the successful amplification result of a mutation by the adaptor ligation method of the present invention targeting a template including a KRAS G12V mutation in one embodiment of the present invention.
  • Figure 8 illustrates the mutation-specific 5' adapter ligation process of the present invention.
  • the NGS preprocessing process refers to the process of processing sample DNA or RNA so that it can be recognized by the NGS equipment and subjected to base sequence analysis.
  • the sample DNA or RNA is usually cut into an appropriate size, and then an adapter oligonucleotide with a specific base sequence is attached to both ends.
  • the product created in this way is called a library.
  • library amplification is performed via end repair, 3’ terminal adenine addition, adapter attachment, and PCR amplification.
  • Terminal repair In many cases, the terminal portion of a nucleic acid that has completed segmentation does not form a complete duplex structure. These incomplete terminal portions are cut off using DNA polymerase or filled in with complementary bases to form a complete duplex structure all the way to the end. At this time, an additional phosphate group is added to the 5'-end.
  • An adapter is an oligonucleotide with a unique base sequence that can be recognized by NGS equipment.
  • the process of attaching the adapter to DNA uses a ligase enzyme.
  • Adapters are usually in a Y shape (forked) or U shape (hairpin) and have thymine attached to the 3'-terminal so that they can be easily attached to the A-terminal product by the ligase enzyme.
  • Adapters have a unique base sequence that can distinguish samples, and this is called an index or barcode.
  • the sample index usually consists of several to several dozen base sequences, and each equipment reads the agreed-upon index base sequence and classifies which sample the corresponding library DNA originated from.
  • the library is amplified through the PCR process.
  • an end repair process is performed to phosphorylate the 5' end of the template and hydroxylate the 3' end.
  • a blunt end adapter ligation process is performed.
  • the adapter used here has its 3' end phosphorylated and does not form an adapter dimer, so it does not cause an adapter dimer problem in the subsequent NGS analysis process.
  • a product is created in which the adapter sequence is linked only to the 3' portion of the template (see Figure 7).
  • TS probe target specific probe
  • U probe universal probe
  • a Lambda nuclease treatment process is performed before the PCR amplification process.
  • the wild-type molecule and probes with 5' phosphorylation modification are degraded by the enzymatic action, leaving only the mutant molecules.
  • only the mutant molecules with the adapter sequence on both sides are amplified by the PCR process, and mutant enrichment is achieved.
  • the present invention can provide a probe that complementarily binds to a target DNA having a mutant base, wherein the probe includes a base sequence complementary to the base sequence of the target DNA, but is characterized in that the base at the second position away from the mutant base position is replaced with an artificial mismatch base.
  • the mismatch base refers to a mismatch base with respect to a base located at the second position downstream (from 5' to 3' of the target DNA base sequence to be detected) based on the position of the mutant base present in the target DNA (position 0). More specifically, it refers to a non-complementary (mismatched base) base with respect to a base located at the second position downstream with the position of the mutant base present in the target DNA as the reference position 0.
  • the probe provided in the present invention corresponds to a target-specific probe, and includes a base sequence region complementary to target DNA for binding to the target DNA; and a base sequence region complementary to a universal probe base sequence for binding to the universal probe; wherein, in the base sequence complementary to the target DNA, the second base located downstream from a mutant base present in the target DNA as the reference 0 position is substituted with a mismatched base.
  • the target DNA to be detected using the probe of the present invention may be cfDNA (cell free DNA) as a mutation having a low gene frequency, and the cfDNA (cell free DNA) may preferably be a nucleic acid fragment occurring at the time of disease onset.
  • the present invention can provide a mutant DNA-specific adapter ligation method, which includes a step of hybridizing the probe and universal probe of the present invention with target DNA and then reacting them by treating them with FEN1 (flap endonuclease 1) and HIFI ligase.
  • the present invention can provide a method for enriching mutant target DNA, including a step of hybridizing the probe and universal probe of the present invention with target DNA, treating with FEN1 (flap endonuclease 1) and HIFI ligase to perform adapter ligation, and then performing a PCR amplification reaction using a universal primer.
  • FEN1 overlap endonuclease 1
  • HIFI ligase HIFI ligase
  • the present invention can improve the difficulty of analyzing samples with low sensitivity or low VAF due to high cost, which are problems of existing nucleic acid analysis technologies, and can specifically ligate adapters only to mutant molecules by using FEN1 and HIFI ligases and two types of probes.
  • efficient and successful mutant enrichment was achieved by performing a PCR process using a universal primer after adapter ligation.
  • This method of the present invention can be useful not only for disease diagnosis based on liquid biopsy, but also for mutation detection with a reduced risk of false positives.
  • TS probe name Sequence (5' - 3') Length (nt) Sequence number TS probe 2 GGCACTCTTGCCTACGCCA A CAATGCGCGACATTCCGAAG 40 1 TS probe 2(3) GGCACTCTTGCCTACGCC GA CAATGCGCGACATTCCGAAG 40 2 TS probe 2(4) GGCACTCTTGCCTACGC T A A CAATGCGCGACATTCCGAAG 40 3 TS probe 2(5) GGCACTCTTGCCTACG T CA A CAATGCGCGACATTCCGAAG 40 4
  • the base of the complementary TS probe for the mutant base is indicated in bold blue, which is "A" located at the 20th position in the 5'->3' direction in each probe sequence.
  • the second base downstream from the mutation site (in the 5' to 3' direction of the detection target DNA sequence) was selected as the final mismatch site.
  • TS probe sequence name Sequence (5' - 3') Length (nt) order number KRAS G12V TS probe /5Phos/GGCACTCTTGCCTACGC G A A C AATGCCGACATTCCGAAGAAAAA/3ddC/ 45 5 EGFR L858R TS probe /5Phos/CCCAGCAGTTTGG G C C G AATGCGCGACATTCCGAAGAAAAA/3ddC/ 41 6 EGFR T790M TS probe /5Phos/AGCCGAAGGGCATGAGCT T C A T AATGCGCGACATTCCGAAGAAAAA/3ddC/ 46 7 KRAS G12D TS probe /5Phos/GGCACTCTTGCCTACGC G A T C AATGCCGACATTCCGAAGAAAAA/3ddC/ 45 8 NRAS Q61K TS probe /5Phos/ATTGGTCTCTCATGGCACTGTACTCTTC C T T T AATGCGCGACATTCCGAAGAAAAA/3ddC
  • the base of the complementary TS probe for the mutant base is indicated in bold blue, and is the second base located in the 5'->3' direction from the mismatch position base of each probe (0th).
  • KRAS G12V TS probe it is bold “A”
  • EGFR L858R TS probe it is bold “C”
  • EGFR T790M TS probe it is bold “A”
  • KRAS G12D TS probe it is bold "T”
  • NRAS Q61K TS probe it is bold "T”
  • NRAS A59T TS probe it is bold "T”
  • PIK3CA E545K TS probe it is bold "T”.
  • the region binding to the TS probe was referenced from the bacterial 16s rRNA gene sequence to suppress unintended structure generation when applied to cfDNA samples in the future.
  • the portion binding to the template was referenced from the NGS sequencing primer sequence and was designed so that it could be used for PCR using universal primers thereafter, and a universal probe consisting of the sequences in Table 2 below was designed.
  • the basic adapter sequence was referenced from the NGS Y-shaped adapter. To suppress the formation of adapter dimers during the adapter ligation process, one strand was 3' phosphorylated, and the other strand was 5' phosphorylated so that it could be linked to the template using a ligase.
  • the adapter sequence is as shown in Table 4 below.
  • Adapter sequence name Sequence (5' - 3') Length (nt) order number 3’ blocked Adapter-a /5Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACATCACGATCTCGTATGCCGTCTTCTGCTTG/3ddC/ 63 16 3’ blocked Adapter-b GCTCTTCCGATCT/3Phos/ 13 17
  • the buffer is HIFI Taq DNA It was found that using Ligase buffer was appropriate.
  • the present invention aims to be applied to various mutation targets. To this end, diversity was provided to the 3' terminal sequence of the U probe, which is affected by the mutation position sequence of the target DNA.
  • the inventors of the present invention were able to find out that when a probe containing an artificial mismatch according to the present invention is used, mutation-specific adapter ligation can be effectively performed, thereby successfully amplifying and detecting a mutation target to be detected.

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Abstract

La présente invention concerne un procédé de ligature d'adaptateur spécifique d'un mutant à haut rendement et, en particulier : une sonde se liant de manière complémentaire à un ADN cible ayant une base mutante, la sonde comprenant une séquence de base complémentaire de la séquence de base de l'ADN cible, une base en position 2 à partir de la base mutante définie comme position 0 étant substituée par une base de mésappariement artificiel ; une composition pour capturer un ADN cible mutant pour une analyse de séquençage de nouvelle génération (NGS), comprenant la sonde ; un procédé de ligature d'adaptateur spécifique à l'ADN mutant comprenant les étapes consistant à hybrider la sonde et une sonde universelle avec un ADN cible, puis à traiter celle-ci avec une endonucléase à clapet 1 (FEN1) et une ligase HIFI pour provoquer une réaction entre celles-ci ; et un procédé d'enrichissement d'un ADN cible mutant, comprenant les étapes consistant à hybrider la sonde et une sonde universelle avec un ADN cible, puis à traiter celle-ci avec FEN1 et une ligase HIFI pour effectuer une ligature d'adaptateur, puis à effectuer une réaction d'amplification par PCR à l'aide d'amorces universelles.
PCT/KR2024/015269 2023-10-11 2024-10-08 Procédé de ligature d'adaptateur spécifique d'un mutant à haut rendement Pending WO2025079958A1 (fr)

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KR20230135160 2023-10-11
KR10-2023-0135160 2023-10-11
KR10-2024-0134708 2024-10-04
KR1020240134708A KR20250052964A (ko) 2023-10-11 2024-10-04 높은 처리량의 돌연변이 특이적 아답터 리게이션 방법

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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR100993349B1 (ko) * 2007-02-23 2010-11-10 주식회사 파나진 지지체에 고정된 ｐｎａ 프로브와 표적핵산의 혼성화 효율또는 특이도를 증가시키기 위한 방법, 조성물 및 키트
KR20150067161A (ko) * 2012-09-04 2015-06-17 가던트 헬쓰, 인크. 희귀 돌연변이 및 카피수 변이를 검출하기 위한 시스템 및 방법
KR101550489B1 (ko) * 2010-03-08 2015-09-07 다나-파버 캔서 인스티튜트 인크. 기준 차단 서열을 이용한 완전 ｃｏｌｄ－ｐｃｒ 풍부화
US20210155980A1 (en) * 2015-09-25 2021-05-27 Canexia Health Inc. Molecular quality assurance methods for use in sequencing
KR102275038B1 (ko) * 2019-12-11 2021-07-08 (주) 제노텍 대립 유전자의 구분성을 높이는 pcr 방법 및 pcr 킷트
US11773434B2 (en) * 2017-06-20 2023-10-03 The Medical College Of Wisconsin, Inc. Assessing transplant complication risk with total cell-free DNA

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR100993349B1 (ko) * 2007-02-23 2010-11-10 주식회사 파나진 지지체에 고정된 ｐｎａ 프로브와 표적핵산의 혼성화 효율또는 특이도를 증가시키기 위한 방법, 조성물 및 키트
KR101550489B1 (ko) * 2010-03-08 2015-09-07 다나-파버 캔서 인스티튜트 인크. 기준 차단 서열을 이용한 완전 ｃｏｌｄ－ｐｃｒ 풍부화
KR20150067161A (ko) * 2012-09-04 2015-06-17 가던트 헬쓰, 인크. 희귀 돌연변이 및 카피수 변이를 검출하기 위한 시스템 및 방법
US20210155980A1 (en) * 2015-09-25 2021-05-27 Canexia Health Inc. Molecular quality assurance methods for use in sequencing
US11773434B2 (en) * 2017-06-20 2023-10-03 The Medical College Of Wisconsin, Inc. Assessing transplant complication risk with total cell-free DNA
KR102275038B1 (ko) * 2019-12-11 2021-07-08 (주) 제노텍 대립 유전자의 구분성을 높이는 pcr 방법 및 pcr 킷트

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