WO2019140298A1 - Novel primers and uses thereof - Google Patents

Novel primers and uses thereof Download PDF

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Publication number
WO2019140298A1
WO2019140298A1 PCT/US2019/013346 US2019013346W WO2019140298A1 WO 2019140298 A1 WO2019140298 A1 WO 2019140298A1 US 2019013346 W US2019013346 W US 2019013346W WO 2019140298 A1 WO2019140298 A1 WO 2019140298A1
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WO
WIPO (PCT)
Prior art keywords
section
target
adaptor
specific
stem
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US2019/013346
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English (en)
French (fr)
Inventor
Bernhard Zimmermann
Ryan Swenerton
Fei Lu
Scott DASHNER
Himanshu SETHI
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Natera Inc
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Natera Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority to EP19702785.7A priority Critical patent/EP3737773B1/en
Priority to EP25214405.0A priority patent/EP4671380A3/en
Priority to CA3088891A priority patent/CA3088891C/en
Priority to CN201980008237.5A priority patent/CN111699269B/zh
Priority to US16/959,949 priority patent/US20210071246A1/en
Priority to JP2020558864A priority patent/JP7322063B2/ja
Application filed by Natera Inc filed Critical Natera Inc
Priority to CN202511678455.3A priority patent/CN121653234A/zh
Priority to EP23167431.8A priority patent/EP4245863B1/en
Publication of WO2019140298A1 publication Critical patent/WO2019140298A1/en
Anticipated expiration legal-status Critical
Priority to JP2023121655A priority patent/JP7773511B2/ja
Priority to JP2025188258A priority patent/JP2026016789A/ja
Ceased legal-status Critical Current

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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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6853—Nucleic acid amplification reactions using modified primers or templates
    • 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
    • C12Q1/6858—Allele-specific amplification
    • 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
    • C12Q2525/00—Reactions involving modified oligonucleotides, nucleic acids, or nucleotides
    • C12Q2525/10—Modifications characterised by
    • C12Q2525/155—Modifications characterised by incorporating/generating a new priming site
    • 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
    • C12Q2525/00—Reactions involving modified oligonucleotides, nucleic acids, or nucleotides
    • C12Q2525/10—Modifications characterised by
    • C12Q2525/161—Modifications characterised by incorporating target specific and non-target specific sites
    • 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
    • C12Q2525/00—Reactions involving modified oligonucleotides, nucleic acids, or nucleotides
    • C12Q2525/10—Modifications characterised by
    • C12Q2525/191—Modifications characterised by incorporating an adaptor
    • 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
    • C12Q2525/00—Reactions involving modified oligonucleotides, nucleic acids, or nucleotides
    • C12Q2525/30—Oligonucleotides characterised by their secondary structure
    • C12Q2525/301—Hairpin oligonucleotides
    • 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
    • C12Q2537/00—Reactions characterised by the reaction format or use of a specific feature
    • C12Q2537/10—Reactions characterised by the reaction format or use of a specific feature the purpose or use of
    • C12Q2537/143—Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
    • 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
    • C12Q2563/00—Nucleic acid detection characterized by the use of physical, structural and functional properties
    • C12Q2563/179—Nucleic acid detection characterized by the use of physical, structural and functional properties the label being a nucleic acid

Definitions

  • the present inventions are directed to compositions, methods, and kits for amplification of nucleic acids.
  • the inventions described herein relate to a composition comprising a primer that is: (a) a loopable primer comprising a target- specific section, an adaptor section, and a stem-forming section, wherein the stem-forming section is hybridizable to a portion of the target- specific section to form a stem structure, or (b) a split primer comprising a first target- specific section, a second target- specific section, and an adaptor section positioned between the first target- specific section and the second target- specific section, or (c) a split-loopable primer comprising a first target- specific section, a second target- specific section, and a stem-forming section positioned between the first target- specific section and the second target- specific section, and an adaptor section, or comprising a first adaptor section, a second adaptor section, and a stem-forming section positioned between the first adaptor section and the second
  • the inventions described herein relate to a method for determining copy number variation of a target locus of interest, comprising: pre-amplifying the target locus of interest from a template DNA using at least two pre-amplification cycles with: (a) one or more loopable primers each comprising a target- specific section, an adaptor section, a molecular indexing section, and a stem-forming section, wherein the target- specific section comprises a 5’- portion and a 3’-portion and the stem-forming section is hybridizable to the 3’-portion of the target- specific section to form a stem structure and a loop comprising the adaptor section and the molecular indexing section and the 5’-portion of the target- specific section, wherein the adaptor section comprises a universal adaptor sequence for PCR amplification, and wherein the molecular indexing section comprises a molecule indexing sequence, (b) one or more split- loopable primers each comprising a first target- specific section, a second target
  • the inventions described herein relate to a method for determining fetal aneuploidy, comprising: pre- amplifying a plurality of target loci of interest of one or more chromosomes from cell-free DNA isolated from a maternal blood sample, using at least two pre amplification cycles with: (a) a plurality of loopable primers each comprising a target- specific section, an adaptor section, a molecular indexing section, and a stem-forming section, wherein the target- specific section comprises a 5’-portion and a 3’-portion and the stem- forming section is hybridizable to the 3’-portion of the target- specific section to form a stem structure and a loop comprising the adaptor section and the molecular indexing section and the 5’-portion of the target- specific section, wherein the adaptor section comprises a universal adaptor sequence for PCR amplification, and wherein the molecular indexing section comprises a molecule indexing sequence, (b) a plurality of loopable primers each comprising
  • amplification at least a first split-loopable primer and a second split-loopable primer each comprising a first target- specific section, a second target- specific section, a stem- forming section positioned between the first target- specific section and the second target- specific section, and an adaptor section, wherein the stem-forming section is hybridizable to a portion of the second target- specific section to form a stem structure and a loop comprising the adaptor section, wherein the adaptor section comprises a universal adaptor sequence for PCR amplification, or (c) at least a first split-loopable primer and a second split-loopable primer each comprising a first adaptor section, a second adaptor section, a stem-forming section positioned between the first adaptor section and the second adaptor section, and a target- specific section, wherein the target- specific section comprises a 5’-portion and a 3’-portion and the stem- forming section is hybridizable to the 3’-portion of the target- specific section to form
  • the inventions described herein relate to a method for allele- specific digital PCR (dPCR), comprising: pre-amplifying one or more target loci of interest from a template DNA using at least two pre-amplification cycles with: (a) at least a first loopable primer and a second loopable primer each comprising a target- specific section, an adaptor section, and a stem- forming section, wherein the target- specific section comprises a 5’-portion and a 3’-portion and the stem- forming section is hybridizable to the 3’-portion of the target- specific section to form a stem structure and a loop comprising the adaptor section and the 5’-portion of the target- specific section, wherein the adaptor section of the first loopable primer comprises a universal adaptor sequence for PCR amplification and a first probe- specific sequence capable of binding to a first fluorescent probe, wherein the adaptor section of the second loopable primer comprises a universal adaptor sequence for PCR amplification and a second probe-
  • dPCR allele- specific
  • FIG. 1 shows some embodiments of the primers described herein (Scheme A to C and Control Scheme).
  • FIG. 5 shows workflow of an example amplification process including 2 pre amplification cycles using the primers described herein.
  • FIG. 10 shows primer and product sequences according to one embodiment of a loopable primer, which includes 2 mismatched nt in the loopable primer.
  • the loopable primer according to Scheme B has a preferred annealing temperature and a melting temperature for PCR reaction, wherein the stem-forming section and the 5’-portion of the target- specific section form the stem structure at the preferred annealing temperature or below and do not form the stem structure at the melting temperature or above.
  • the preferred annealing temperature is 60°C or less, 59°C or less, or 58°C or less, or 57°C or less, or 56°C or less, or 55°C or less, 54°C or less, or 53°C or less, or 52°C or less, or 5l°C or less, or 50°C or less.
  • Scheme C comprises, from 5’ to 3’, first target- specific section, an adaptor section, a molecular indexing section, second target- specific section.
  • the split-loopable primer further comprises a molecular indexing section comprising a molecule indexing sequence.
  • the molecular indexing section can be, for example, positioned between the adaptor section and the (second) target- specific sections.
  • the molecular indexing section can be, for example, positioned at 3’ side of the (second) adaptor section.
  • the length of each molecular indexing sequence is about 1-20 bp, or about 2-15 bp, or about 3-10 bp, or about 4-8 bp.
  • a preferred embodiment of the split-loopable primer according to Scheme D comprises, from 5’ to 3’, first target- specific section, one or more mismatched nucleotides, a stem-forming section, an adaptor section, a molecular indexing section, and second target- specific section, wherein the stem-forming section is the reverse complement of part of the second target- specific section.
  • the size of the stem structure formed between the stem forming section and the 3’-portion of the target- specific section is about 5-20 bp, or about 5-10 bp, or about 10-15 bp, or about 15-20 bp.
  • the composition comprises at least 50, at least 100, at least 200, at least 500, at least 1,000, at least 2,000, at least 5,000, or at least 10,000 different loopable primers each comprising a different stem-forming section. In some embodiments, the composition comprises at least 50, at least 100, at least 200, at least 500, at least 1,000, at least 2,000, at least 5,000, or at least 10,000 different loopable primers each comprising a different molecular indexing sequence.
  • the composition comprises at least 50, at least 100, at least 200, at least 500, at least 1,000, at least 2,000, at least 5,000, or at least 10,000 different split- loopable primers each comprising a different stem-forming section. In some embodiments, the composition comprises at least 50, at least 100, at least 200, at least 500, at least 1,000, at least 2,000, at least 5,000, or at least 10,000 different split- loopable primers each comprising a different molecular indexing sequence.
  • the PCR primer comprises a sequencing adaptor for downstream high-throughput sequencing of the PCR products.
  • the PCR primer comprises a sample barcode for pooling of the PCR products for further analysis.
  • the split-loopable primer according to Scheme D has a preferred annealing temperature for PCR reaction and a melting temperature, wherein the stem forming section and the second target- specific section form the stem structure at the preferred annealing temperature or below and do not form the stem structure at the melting temperature or above, and wherein the annealing temperature for the pre-amplification cycles is at or below the preferred annealing temperature (e.g., 60°C or less, 59°C or less, or 58°C or less, or 57°C or less, or 56°C or less, or 55°C or less, 54°C or less, or 53°C or less, or 52°C or less, or 5l°C or less, or 50°C or less).
  • the preferred annealing temperature for PCR reaction and a melting temperature
  • the stem forming section and the second target- specific section form the stem structure at the preferred annealing temperature or below and do not form the stem structure at the melting temperature or above
  • kits for amplifying a target locus of interest from a template DNA comprising a loopable primer described above or a split primer described above or a split-loopable primer described above.
  • the loopable primer of Scheme A (3’-target-StemLoop) that hides/protects the universal adapter sequences and the MIT sequences improves assay specificity by suppressing primer dimers and non-specific binding in high multiplex PCR. Accordingly, the loopable primer of Scheme A is particularly useful for the following applications:

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  • Chemical & Material Sciences (AREA)
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  • Organic Chemistry (AREA)
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  • Bioinformatics & Cheminformatics (AREA)
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  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
PCT/US2019/013346 2018-01-12 2019-01-11 Novel primers and uses thereof Ceased WO2019140298A1 (en)

Priority Applications (10)

Application Number Priority Date Filing Date Title
CN202511678455.3A CN121653234A (zh) 2018-01-12 2019-01-11 新引物及其用途
EP25214405.0A EP4671380A3 (en) 2018-01-12 2019-01-11 Novel primers and uses thereof
CA3088891A CA3088891C (en) 2018-01-12 2019-01-11 Novel primers and uses thereof
CN201980008237.5A CN111699269B (zh) 2018-01-12 2019-01-11 新引物及其用途
US16/959,949 US20210071246A1 (en) 2018-01-12 2019-01-11 Novel primers and uses thereof
EP19702785.7A EP3737773B1 (en) 2018-01-12 2019-01-11 Novel primers and uses thereof
EP23167431.8A EP4245863B1 (en) 2018-01-12 2019-01-11 Novel primers and uses thereof
JP2020558864A JP7322063B2 (ja) 2018-01-12 2019-01-11 新規なプライマーおよびその使用
JP2023121655A JP7773511B2 (ja) 2018-01-12 2023-07-26 新規なプライマーおよびその使用
JP2025188258A JP2026016789A (ja) 2018-01-12 2025-11-07 新規なプライマーおよびその使用

Applications Claiming Priority (2)

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US201862617066P 2018-01-12 2018-01-12
US62/617,066 2018-01-12

Publications (1)

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WO2019140298A1 true WO2019140298A1 (en) 2019-07-18

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EP (3) EP4245863B1 (https=)
JP (3) JP7322063B2 (https=)
CN (2) CN111699269B (https=)
CA (2) CA3088891C (https=)
WO (1) WO2019140298A1 (https=)

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