EP3610031A1 - Quantification et qualification de banques - Google Patents

Quantification et qualification de banques

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Publication number
EP3610031A1
EP3610031A1 EP17905299.8A EP17905299A EP3610031A1 EP 3610031 A1 EP3610031 A1 EP 3610031A1 EP 17905299 A EP17905299 A EP 17905299A EP 3610031 A1 EP3610031 A1 EP 3610031A1
Authority
EP
European Patent Office
Prior art keywords
dna fragments
amplified dna
amplified
primers
primer
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.)
Pending
Application number
EP17905299.8A
Other languages
German (de)
English (en)
Other versions
EP3610031A4 (fr
Inventor
Douglas A. Amorese
Bin Li
Benjamin G. Schroeder
Richard A. FEKETE
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.)
Tecan Genomics Inc
Original Assignee
Nugen Technologies 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
Application filed by Nugen Technologies Inc filed Critical Nugen Technologies Inc
Publication of EP3610031A1 publication Critical patent/EP3610031A1/fr
Publication of EP3610031A4 publication Critical patent/EP3610031A4/fr
Pending legal-status Critical Current

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Classifications

    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B40/00—ICT specially adapted for biostatistics; ICT specially adapted for bioinformatics-related machine learning or data mining, e.g. knowledge discovery or pattern finding
    • G16B40/10—Signal processing, e.g. from mass spectrometry [MS] or from PCR
    • 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
    • C12Q1/6851—Quantitative amplification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • 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
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B25/00—ICT specially adapted for hybridisation; ICT specially adapted for gene or protein expression
    • G16B25/20—Polymerase chain reaction [PCR]; Primer or probe design; Probe optimisation

Definitions

  • Next Gen DNA sequencers normally use DNA fragments having ends of the known sequence. Having known sequence on both ends enables the DNA fragments to be amplified, immobilized and offers a start position for sequencing (e.g., a priming site).
  • the ends of known sequence are typically referred to as adapters; they adapt the DNA fragment to the needs of the sequencer. Not all DNA fragments in a solution have adapters present on each end for sequence determination.
  • a PCR amplification step using two distinct primers, each specific to one of the adapters, is typically employed to enrich for fragments with two different adapters on their ends.
  • This collection of DNA fragments with adapters on the ends is typically referred to as a library.
  • These libraries may be immobilized on a solid support such that a spatial distance between library elements (e.g., adapters with different DNA fragments inserted between them) allows for visualization (detection) and recognition of single elements from each other.
  • the cluster is homogeneous and does not contain DNA from any other library elements. If clusters are too close together or in the extreme, overlapping, image analysis software may have difficulty distinguishing the boundaries of the clusters and combine them into a single feature for data extraction. Since data from this cluster is derived from two different DNA fragments with two different sequences, the software may not be able to determine the sequences accurately. If clusters are further apart, each cluster can be analyzed separately and the sequence accurately determined. If clusters are too far apart, the sequencing becomes inefficient. The cost of processing a sample is fixed, and the cost per cluster increases.
  • the method may include providing DNA fragments, amplifying the DNA fragments by polymerase chain reaction (PCR) in the presence of primers each labeled with a fluorophore. In these instances, only a predetermined number of fluorophores are attached to each DNA fragment.
  • the method may further include detecting a fluorescent signal produced by the amplified DNA fragments and calculating a number of the amplified DNA fragments based on the detected fluorescent signal.
  • the method may further include removing primers that are not incorporated into the amplified DNA fragments or quenching the signal produced by the primers that are not incorporated into the amplified DNA fragments.
  • the method may further include the step of the fluorescence-based sequencing of the amplified DNA fragments.
  • the signal produced by the amplified DNA fragments may be detected by detecting the fluorescent signal produced by fluorophores incorporated into the amplified DNA fragments using a fluorometer.
  • the method may further include generating a standard curve indicating a relationship between the number of DNA fragments derived from a standard library and fluorescent signals produced by the DNA fragments.
  • the number of the amplified DNA fragments may be calculated based on the detected signal by calculating the number of the amplified DNA fragments based on the detected fluorescent signal and the standard curve.
  • the method may further include diluting the amplified DNA fragments to a predetermined concentration suitable for the fluorescence-based sequencing.
  • the method may further include determining a characteristic of the amplified DNA fragments.
  • the characteristic of the amplified DNA fragments may include an average size of the amplified DNA fragment.
  • the DNA fragments may include an adapter, and the primers are complementary to the adapter.
  • Some embodiments relate to a nucleic acid library including DNA fragments each attached with only a predetermined number of fluorophores such that a number of the DNA fragments is calculated based on the fluorescent signal produced by the attached DNA fragments.
  • the DNA fragments are PCR amplicons that are generated using primers each labeled with a fluorophore such that with only a predetermined number of fluorophores are attached to each PCR amplicon fragment.
  • the DNA fragments may include an adapter, and the primers are complementary to the adapter.
  • the method may include generating DNA fragments using the DNA sample and amplifying the DNA fragments by polymerase chain reaction (PCR) in the presence of primers each labeled with a fluorophore. In these instances, only a predetermined number of fluorophores are attached to each DNA fragment.
  • the method may further include detecting a fluorescent signal produced by the amplified DNA fragments, calculating a number of the amplified DNA fragments based on the detected fluorescent signal, diluting the amplified DNA fragments to a predetermined concentration suitable for the fluorescence-based sequencing, and sequencing at least one portion of the amplified DNA fragments using fluorescence- based sequencing techniques.
  • the method may further include removing primers that are not incorporated into the amplified DNA fragments or quenching the signal produced by the primers that are not incorporated into the amplified DNA fragments.
  • only a single fluorophore is attached to each DNA fragment.
  • the signal produced by the amplified DNA fragments may be detected by detecting the fluorescent signal produced by fluorophores incorporated into the amplified DNA fragments using a fluorometer.
  • the method may further include generating a standard curve indicating a relationship between the number of DNA fragments derived from a standard library and fluorescent signals produced by the DNA fragments.
  • the number of the amplified DNA fragments based on the detected signal may be calculated by calculating the number of the amplified DNA fragments based on the detected fluorescent signal and the standard curve.
  • the method may further include determining a characteristic of the amplified DNA fragments.
  • the characteristic of the amplified DNA fragments may include an average size of the amplified DNA fragment.
  • the DNA fragments may include an adapter, and the primers are complementary to the adapter.
  • Some embodiments may further include a kit including adapters capable of linking to DNA fragments and primers complementary to the adapter.
  • Each primer may be labeled with a fluorophore such that the DNA fragments are amplified using the primers to attach each DNA fragment with only a predetermined number of fluorophores.
  • the kit may include one or more polymerases.
  • the kit may include reagents for amplification.
  • the kit may include reagents for sequencing.
  • the kit may include written instructions for the use of the kit.
  • the kit may include dATP, dCTP, dGTP, dTTP, or any mixture thereof.
  • FIG. 1 shows an example of library quantitation.
  • FIG. 2 shows another example of library quantitation.
  • FIG. 3 shows yet another example of library quantitation.
  • Embodiments of the present disclosure relate to a surprising discovery that attaching a fluorescent dye to DNA fragments for library quantitation and qualification does not interfere with subsequent sequence determination.
  • primers attached fluorescent dye are used for library quantitation and qualification. While the primers attached fluorescent dye remain in the library, the subsequent sequencing of the library may be implemented using a fluorescence-based sequencing techniques.
  • Still others use qPCR to more accurately determine the mass of actual library (not total nucleic acid) and use this in combination with fragment sizes from the BioAnalyzer to get a more accurate determination of the number of cluster forming units and how to appropriately dilute the sample to get the desired concentration for applying to the DNA sequencer.
  • the present disclosure provides techniques for determining the number of elements capable of generating clusters. Some embodiments of the present disclosure relate to a method for library quantitation and qualification without relying on techniques such as UV spectrophotometry, qPCR, and average fragment size estimation.
  • a fluorescent-labeled PCR primer may be employed in the library enrichment step. Since the enrichment step produces amplicons with a single fluorophore or a predetermined number of fluorophores per fragment, the number of molecules may be determined. Each amplification product (e.g., amplicon) may have a predetermined number of fluorophores independent of its length. For example, only those elements which have been amplified have a fluorophore bound. Following PCR enrichment, unincorporated primers are removed, and the amount of fluorescent primer/amplicon is determined fluorometrically. A standard curve may be generated to determine the number of fluorescent fragments in the solution.
  • amplicon e.g., amplicon
  • the term "adaptor,” as used herein, can refer to an oligonucleotide of known sequence, the attachment of which to a specific nucleic acid sequence or a target polynucleotide strand of interest enables the generation of amplification- ready products of the specific nucleic acid or the target polynucleotide strand of interest.
  • the specific nucleic acid samples can be fragmented or not prior to the addition of at least one adaptor.
  • Adaptor designs are envisioned which are suitable for generation of amplification-ready products of specific sequence regions/strands of interest.
  • the two strands of the adaptor can be self-complementary, noncomplementary or partially complementary.
  • Adaptors can contain at least a partial forward sequence priming site and a random sequence.
  • the terms “amplifying,” “amplification” and to “amplify” a specific nucleic acid as used herein can refer to a procedure wherein multiple copies of the nucleic acid sample of interest are generated, for example, in the form of DNA copies. Many methods and protocols are known in the art to amplify nucleic acids, such as PCR and qPCR.
  • the term “cDNA” as used herein can refer to complementary DNA. The DNA can be synthesized in a reaction catalyzed by reverse transcriptase and DNA polymerase from a messenger RNA (mRNA) template.
  • mRNA messenger RNA
  • the term "complementary" as used herein can refer to complementarity to all or only to a portion of a sequence.
  • the number of nucleotides in a hybridizable sequence of a specific oligonucleotide primer or probe can be such that stringency conditions used to hybridize the oligonucleotide primer or probe can prevent excessive random non-specific hybridization.
  • the number of nucleotides in the hybridizing portion of the oligonucleotide primer or probe can be at least as great as the defined sequence of the target polynucleotide that the oligonucleotide primer or probe hybridizes to usually about 20 to about 50 nucleotides.
  • the target polynucleotide/oligonucleotide can be larger than the oligonucleotide primer, primers or probe.
  • the term "denaturing" as used herein can refer to the separation of double-stranded nucleic acid into single strands. Denaturation can be achieved using any of the methods known in the art including, but not limited to, physical, thermal, and/or chemical denaturation.
  • genomic DNA can refer to chromosomal DNA, abbreviated as gDNA for genomic deoxyribonucleic acid.
  • gDNA includes the genetic material of an organism.
  • the term "genome” as used herein can refer to sequences, either DNA, RNA or cDNA derived from a patient, a tissue, an organ, a single cell, a tumor, a specimen of an organic fluid taken from a patient, freely circulating nucleic acid, a fungus, a prokaryotic organism and a virus.
  • RNAscriptome can be all RNA sequences that can reflect a partial or entire expressed genome of an organism.
  • kit can refer to any system for delivering materials.
  • delivery systems can include elements allowing the storage, transport, or delivery of reaction components such as oligonucleotides, buffering components, additives, reaction enhancers, enzymes and the like in the appropriate containers from one location to another commonly provided with written instructions for performing the assay.
  • Kits can include one or more enclosures or boxes containing the relevant reaction reagents and supporting materials.
  • the kit can may include two or more separate containers wherein each of those containers includes a portion of the total kit components. The containers can be delivered to the intended recipient together or separately.
  • NA-modifying enzyme can refer to a DNA-specific modifying enzyme.
  • the NA-modifying enzyme can be selected for specificity for double-stranded DNA.
  • the enzyme can be a duplex- specific endonuclease, a blunt-end frequent cutter restriction enzyme, or another restriction enzyme.
  • nucleic acid fragment and “specific nucleic acid” are used interchangeably and as used herein, can refer to a portion of a nucleic acid sample.
  • the nucleic acids in the input sample can be fragmented into a population of fragmented nucleic acid molecules or to polynucleotides of one or more specific size range(s).
  • the phrase "specific nucleic acid sequence” or “specific sequence” as used herein, can be a polynucleotide sequence of interest, for which digital measurement and/or quantitation is desired, including but not limited to a nucleic acid fragment.
  • the specific sequence can be known or not known, in terms of its actual sequence.
  • a "template,” as used herein, can be a polynucleotide that contains the specific nucleic acid sequence.
  • the terms "specific sequence,” “specific nucleic acid sequence,” “specific nucleotide sequence,” “regions of interest,” or “sequence of interest” and, variations thereof, are used interchangeably.
  • the phrases "qualified nucleic acid” and “qualifies the target nucleic acid fragment” as used herein, can refer to a fragment of a gDNA or RNA sequence that is: i.) an acceptable template for a DNA polymerase, i.e.
  • the template can be free of cross-links or inhibitors to the DNA polymerase, or ii.) the template has a modification including, but not limited to, attachment at the 5' and/or 3' end a polynucleotide sequence at least one of a barcode, an adaptor, a sequence complementary to a primer and so on such that the fragment can be modified for purposes of quantitation, amplification, detection or to other methods known to one of skill in the art of gDNA and cDNA sequence analyses.
  • oligonucleotide can refer to a polynucleotide chain, typically less than 200 residues long, e.g., between 15 and 100 nucleotides long, but can also encompass longer polynucleotide chains. Oligonucleotides can be single- or double-stranded. As used in this disclosure, the term “oligonucleotide” can be used interchangeably with the terms “primer,” “probe” and "adaptor.”
  • PCR is an abbreviation of term “polymerase chain reaction,” a commonly available nucleic acids amplification technology.
  • PCR employs two oligonucleotide primers for each strand that are designed such as the extension of one primer provides a template for another primer in the next PCR cycle.
  • Either one of a pair of oligonucleotide primers can be named herein as a “forward” or “reverse” primer with the purpose of distinguishing the oligonucleotide primers in the discussion.
  • a PCR can consist of repetition (or cycles) of (i) a denaturation step which separates the strands of a double stranded nucleic acid, followed by (ii) an annealing step, which allows primers to anneal to positions flanking a sequence of interest; and then (iii) an extension step which extends the primers in a 5' to 3' direction thereby forming a nucleic acid fragment complementary to the target sequence.
  • a denaturation step which separates the strands of a double stranded nucleic acid
  • an annealing step which allows primers to anneal to positions flanking a sequence of interest
  • an extension step which extends the primers in a 5' to 3' direction thereby forming a nucleic acid fragment complementary to the target sequence.
  • Quantitative PCR can refer to a PCR designed to measure the abundance of one or more specific target sequences in a sample. Quantitative measurements can be made using one or more reference nucleic acid sequences that can be assayed separately or together with a target nucleic acid.
  • portion can refer to less than the total length of a nucleic acid sequence, a nucleic acid sequence fragment, a specific nucleic acid sequence, a specific nucleic acid fragment, a probe, a primer and the like.
  • primer can refer to an oligonucleotide, generally with a free 3' hydroxyl group, that can be capable of hybridizing or annealing with a template (such as a specific polynucleotide, target DNA, target RNA, a primer extension product or a probe extension product) and can also be capable of promoting polymerization of a polynucleotide complementary to the template.
  • a primer can contain a non-hybridizing sequence that constitutes a tail of the primer. A primer can hybridize to a target even though its sequences are not fully complementary to the target.
  • the primers utilized herein can be oligonucleotides that are employed in an extension reaction by a polymerase along a polynucleotide template, such as in PCR, qPCR, an extension reaction and the like.
  • the oligonucleotide primer can be a synthetic polynucleotide that can be single stranded, containing a sequence at its 3'- end that can be capable of hybridizing with a sequence of the target polynucleotide.
  • the 3' region of the primer that hybridizes to the specific nucleic acid can may include at least 80%, preferably 90%, more preferably 95%, most preferably 100%, complementarity to a sequence or to a primer binding site.
  • sample can refer to any substance containing or presumed to contain a nucleic acid of interest, and thus includes a sample of nucleic acid, cells, organisms, tissue, fluids (e.g., spinal fluid or lymph fluids), organic fluid taken from a patient, and sample including but not limited to blood, plasma, serum, urine, tears, stool, respiratory and genitourinary tracts, saliva, fragments of different organs, tissue, blood cells, circulating tumor cell (CTC) or a disseminated tumor cell (CTD), bone, samples of in vitro cell cultures or specimens that have been suspected to contain nucleic acid molecules.
  • CTC circulating tumor cell
  • CTD disseminated tumor cell
  • PCR duplicate can refer to any sequencing read that is derived from the same original nucleic acid molecule and so, the same primer/probe extension product sequence, as another sequencing read and is therefore not representative of a unique nucleic acid molecule.
  • the method may include providing DNA fragments and amplifying the DNA fragments by polymerase chain reaction (PCR) in the presence of primers each labeled with a fluorophore. In these instances, only a predetermined number of fluorophores are attached to each DNA fragment.
  • the method may further include detecting a fluorescent signal produced by the amplified DNA fragments and calculating a number of the amplified DNA fragments based on the detected fluorescent signal.
  • Some embodiments relate to a method of library quantitation that uses two or more types of primers.
  • Each primer type can have an associated single fluorophore, multiple fluorophores, or completely lack fluorophores.
  • a first type would have an associated single fluorophore and the second type of primer would lack fluorophores.
  • DNA fragments can be amplified by polymerase chain reaction (PCR) in the presence of at least one primer, with at least one primer labeled with a fluorophore, resulting in a predetermined number of fluorophores being attached to each DNA fragment.
  • PCR polymerase chain reaction
  • a fluorescent signal produced from the amplified DNA fragments is detected, and the number of the amplified DNA fragments based on the detected fluorescent signal is calculated.
  • the DNA fragments can be further prepared for fluorescent sequencing by diluting the amplified DNA fragments to a predetermined concentration.
  • the method may further include removing primers that are not incorporated into the amplified DNA fragments or quenching the signal produced by the primers that are not incorporated into the amplified DNA fragments.
  • the unincorporated fluorescent PCR primers may be removed prior to making a quantitation measurement.
  • the fluorescence of the primers may be quenched.
  • the quenching of unincorporated dye may be achieved by annealing a short oligo, which is complimentary to the fluorescent oligo and has a compound attached capable of quenching the fluorophore.
  • some embodiments of the present disclosure may enable crude samples to be accurately quantitated, mixed in appropriate proportions, and then purified as a collective rather than individually.
  • a separate oligo with a quencher may be used to measure functional elements in a crude mixture is to use an oligo for enrichment that has a hairpin structure and both a fluorophore and quencher.
  • the fluorophore and quencher are in close enough proximity to interfere with fluorescent detection.
  • the spacing between the fluorophore and quencher is increased such that the fluorophore may be detected.
  • the hairpin reforms in oligos that have not been incorporated.
  • the measurement is taken, the oligos incorporated into amplicons are detected, but the unincorporated oligos are dark.
  • only a single fluorophore is attached to each DNA fragment.
  • the method may further include the step of the fluorescence-based sequencing of the amplified DNA fragments.
  • the signal produced by the amplified DNA fragments may be detected by detecting the fluorescent signal produced by fluorophores incorporated into the amplified DNA fragments using a fluorometer (alternatively spelled "fluorimeter").
  • the method may further include generating a standard curve indicating a relationship between DNA fragments derived from a standard library and fluorescent signals produced by the DNA fragments.
  • the number of the amplified DNA fragments may be calculated based on the detected signal by calculating the number of the amplified DNA fragments based on the detected fluorescent signal and the standard curve. [0081] In some embodiments, the method may further include diluting the amplified DNA fragments to a predetermined concentration suitable for the fluorescence-based sequencing.
  • the method may further include determining a characteristic of the amplified DNA fragments. For example, following the measurement of the fluorescent primers, a fluorescent intercalating dye may be added to the sample. The fluorescent intercalating dye may bind proportional to the total mass of double-stranded DNA. The absolute mass may then be determined by comparing this fluorescent reading to a standard curve. With accurate numbers of elements and total mass, the average size of the library fragments may be determined. The measurement may provide data associated with the quality of the library and whether the library is made correctly.
  • the characteristic of the amplified DNA fragments may include an average size of the amplified DNA fragment.
  • the DNA fragments may include an adapter, and the primers are complementary to the adapter.
  • Some embodiments relate to a nucleic acid library including DNA fragments each attached with only a predetermined number of fluorophores such that a number of the DNA fragments is calculated based on the fluorescent signal produced by the attached DNA fragments.
  • the DNA fragments are PCR amplicons that are generated using primers each labeled with a fluorophore such that with only a predetermined number of fluorophores are attached to each PCR amplicon fragment.
  • the DNA fragments may include an adapter, and the primers are complementary to the adapter.
  • the method may include generating DNA fragments using the DNA sample and amplifying the DNA fragments by polymerase chain reaction (PCR) in the presence of primers each labeled with a fluorophore. In these instances, only a predetermined number of fluorophores are attached to each DNA fragment.
  • the method may further include detecting a fluorescent signal produced by the amplified DNA fragments, calculating a number of the amplified DNA fragments based on the detected fluorescent signal, diluting the amplified DNA fragments to a predetermined concentration suitable for the fluorescence-based sequencing, and sequencing at least one portion of the amplified DNA fragments using fluorescence- based sequencing techniques.
  • the method may further include removing primers that are not incorporated into the amplified DNA fragments or quenching the signal produced by the primers that are not incorporated into the amplified DNA fragments.
  • only a single fluorophore is attached to each DNA fragment.
  • the signal produced by the amplified DNA fragments may be detected by detecting the fluorescent signal produced by fluorophores incorporated into the amplified DNA fragments using a fluorometer.
  • the method may further include generating a standard curve indicating a relationship between DNA fragments derived from a standard library and fluorescent signals produced by the DNA fragments.
  • the number of the amplified DNA fragments based on the detected signal may be calculated by calculating the number of the amplified DNA fragments based on the detected fluorescent signal and the standard curve.
  • the method may further include determining a characteristic of the amplified DNA fragments.
  • the characteristic of the amplified DNA fragments may include an average size of the amplified DNA fragment.
  • the DNA fragments may include an adapter, and the primers are complementary to the adapter.
  • kits including adapters capable of linking to DNA fragments and primers complementary to the adapter.
  • Each primer may be labeled with a fluorophore such that the DNA fragments are amplified using the primers to attach each DNA fragment with only a predetermined number of fluorophores.
  • the kit may include one or more polymerases.
  • the kit may include reagents for amplification.
  • the kit may include reagents for sequencing.
  • the kit may include written instructions for the use of the kit.
  • the kit may include dATP, dCTP, dGTP, dTTP, or any mixture thereof.

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Abstract

L'invention concerne des procédés, des compositions et des kits de quantification et de qualification de banques. Dans certains modes de réalisation, l'invention concerne un procédé de quantification de banques. Par exemple, le procédé peut comprendre la préparation de fragments d'ADN, l'amplification des fragments d'ADN par réaction en chaîne par polymérase (PCR) en présence d'amorces, chacune marquée avec un fluorophore. Dans ces cas-là, seul un nombre prédéterminé de fluorophores est fixé à chaque fragment d'ADN. Le procédé peut en outre comprendre la détection d'un signal fluorescent émis par les fragments d'ADN amplifiés et le calcul du nombre de fragments d'ADN amplifiés en fonction du signal fluorescent détecté.
EP17905299.8A 2017-04-11 2017-04-11 Quantification et qualification de banques Pending EP3610031A4 (fr)

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PCT/US2017/027060 WO2018190814A1 (fr) 2017-04-11 2017-04-11 Quantification et qualification de banques

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EP3610031A1 true EP3610031A1 (fr) 2020-02-19
EP3610031A4 EP3610031A4 (fr) 2020-11-18

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US20230069191A1 (en) * 2020-01-31 2023-03-02 Edge Biosystems, Inc. Method for quantitating nucleic acid library

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AU2005295298B2 (en) * 2004-10-18 2011-07-14 Brandeis University Primers, probes and methods for nucleic acid amplification
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WO2018190814A1 (fr) 2018-10-18
JP2020516274A (ja) 2020-06-11

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