[go: up one dir, main page]

WO2015050501A1 - Enrichissement de banques parallèles d'amplification - Google Patents

Enrichissement de banques parallèles d'amplification Download PDF

Info

Publication number
WO2015050501A1
WO2015050501A1 PCT/SG2014/000463 SG2014000463W WO2015050501A1 WO 2015050501 A1 WO2015050501 A1 WO 2015050501A1 SG 2014000463 W SG2014000463 W SG 2014000463W WO 2015050501 A1 WO2015050501 A1 WO 2015050501A1
Authority
WO
WIPO (PCT)
Prior art keywords
dna
polymerase
primer
rna
stranded
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/SG2014/000463
Other languages
English (en)
Inventor
Akshay BHINGE
Jeremie POSCHMANN
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.)
Agency for Science Technology and Research Singapore
Original Assignee
Agency for Science Technology and Research Singapore
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 Agency for Science Technology and Research Singapore filed Critical Agency for Science Technology and Research Singapore
Publication of WO2015050501A1 publication Critical patent/WO2015050501A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • 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

Definitions

  • the present invention relates to the field of molecular biology.
  • the present invention relates to a method for genetic library enrichment using parallel, linear amplification of nucleic acids.
  • Nucleic acid amplification for example DNA amplification, is a key method being used in molecular biology today. It is applied in many fields, from genome mapping, DNA fingerprinting and disease analysis to palaeontology and archaeology. As most nucleic acid extraction techniques result in low concentrations of isolated nucleic acids, amplification methods are applied to increase the concentration of the target nucleic acids post-extraction in order to ensure successful analysis and handling downstream. Many techniques for nucleic amplification have been developed, most importantly the polymerase chain reaction (PCR) in regards to exponential DNA amplification. While the polymerase chain reaction is a commonly used method, it also has its limitations. For example, the presence of contaminating nuclei acid sequences in the same sample could yield false positive results.
  • PCR polymerase chain reaction
  • Chromatin immunoprecipitation is used to isolate DNA from cell lysate, which is then subsequently used in DNA microarrays for further analysis.
  • Chromatin immunoprecipitation generally yields DNA in the nanogram range, which is insufficient for most DNA microarray applications.
  • exponential nucleic acid amplification methods are, for example, random PCR (R-PCR) or ligation-mediated PCR (LM-PCR). While both methods adequately fulfil the pre-requirements for ChlP-chip analysis, these methods also have a number of disadvantages, such as variable amplification fidelity and their inefficient replication of short nucleic acids sequences that are less than 250 base pairs in length.
  • a method of linear amplification of a DNA molecule comprising the steps of:
  • Fig. 1 shows the library size distribution using the Agilent BioAnalyzer.
  • the AmPLE procedure was applied to generate an Illumina library from 50pg of genomic DNA.
  • the figure shows that the library has the recommended size range and is in sufficient amount for Illumina sequencing.
  • Fig. 2 displays the genome browser snapshot of ChlP-Seq signals of libraries generated from different starting amounts.
  • the displayed data indicates H3K27Ac ChlP-Seq performed in GM12878 cell line.
  • Encode indicates external reference data obtained from the ENCODE project.
  • 20M, 5M, 1M indicate the number of cells used for the ChIP in millions.
  • 50pg and lOOpg indicate the ChlP-Seq data generated by applying AmPLE to 50pg and lOOpg of starting ChIP material.
  • ChlP-enriched DNA from the 20M ChIP was serially diluted and used to generate the low input material.
  • Fig. 3 shows a schematic representation of a Y adapted chromatin immunoprecipitation (ChIP) DNA fragment.
  • the Y adapter is made up of two primers, each with a section Al and A2.
  • the Al section of the adaptor is modified to include the T7 promoter sequence in a hairpin, thus ensuring its double-strandedness.
  • the first step shows the Y adapters annealed to each end of a double stranded DNA fragment isolated via chromatin immunoprecipitation.
  • the result of the linear amplification are two identical copies of the target DNA, with integrated T7 promoter sequences at their Al ends
  • These primers are annealed after ligation, a Klenow fragment extension can be performed using the same protocol as mentioned herein.
  • Fig. 4 shows a schematic depiction of the steps required for linear amplification, coupled with the parallel library generation method, as disclosed in the present invention.
  • Acronyms used in the schematic are: dsDNA - double-stranded DNA; dA - single adenosine (A) addition; Klenow - Klenow fragment of E. coli. ; RT-PCR - real-time polymerase chain reaction. DEFINITION OF TERMS
  • nucleic acid refers to a deoxyribonucleotide or ribonucleotide polymer in either single or double stranded form, and unless otherwise limited, encompasses known analogues of natural nucleotides that hybridize to nucleic acids in a manner similar to naturally occurring nucleotides.
  • cDNA refers to complementary DNA, which is a form of nucleic acid sequence that has a base sequence that is complementary to that of a messenger RNA (mRNA) sequence. This means that unlike genomic DNA, complementary DNA contains no intron (i.e. non-coding) sequences
  • amplicon or "amplification product” refers to a nucleic acid fragment, e.g. DNA or RNA, which is the source and/or product of natural or artificial amplification or replication events. It can be formed using various methods, e. g. polymerase chain reactions (PCR), ligase chain reactions (LCR), or natural gene duplication.
  • PCR polymerase chain reactions
  • LCR ligase chain reactions
  • amplification refers to the production of one or more copies of a genetic fragment or target sequence, specifically the amplicon.
  • amplicon is used interchangeably with common laboratory terms, such as PCR product.
  • complementarity refers to two single strands of DNA, in which the nucleotide sequence is such binding will occur as a result of base pairing throughout their entire length. In that case, the complementarity, i.e. the overlap in identity, between the two nucleic acid strands is said to be 100%. Should any gaps or mismatches be present, the complementarity between the two nucleic acid strands could be reduced, depending on the amount of such gaps or mismatches present.
  • the complementarity between two nucleic acid strands can be between 70 to 80%, between 80% to 90%, between 90% to 100%, between 75% to 85%, between 85% to 95%, between 95% to 100%, or between 88% to 98%, or 90%, 95%, 96%, 97%, 98%, 99% or 100%.
  • oligonucleotide refers to short nucleic acid molecules useful, for example, as hybridizing probes, nucleotide array elements or amplification primers. Oligonucleotide molecules are comprised of two or more nucleotides, i.e. deoxyribonucleotides or ribonucleotides, preferably more than five and up to 30 or more. The exact size will depend on many factors, which in turn depend on the ultimate function or use of the oligonucleotide.
  • Oligonucleotides can comprise ligated natural nucleic acid molecules or synthesized nucleic acid molecules and comprise between 10 to 150 nucleotides or between about 12 and about 100 nucleotides which have a nucleotide sequence which can hybridize to a strand of polymorphic DNA, e.g. to permit detection of a polymorphism.
  • Such oligonucleotides may be nucleic acid elements for use on solid arrays (e.g. synthesized or spotted). In some examples of the invention, such oligonucleotides can comprise as few as 20 hybridizing nucleotides, e.g. for assays where the oligonucleotide are used as primers or adapters.
  • the oligonucleotide can comprise as few as about 33 hybridizing nucleotides, e.g. for single base extension assays. Such oligonucleotides may also be primers for use in polymerase chain reaction (PCR) or other reactions.
  • PCR polymerase chain reaction
  • primer refers to a nucleic acid molecule, preferably an oligonucleotide* whether derived from a naturally occurring molecule such as one isolated from a restriction digest or one produced synthetically, which is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product which is complementary to a nucleic acid strand is induced, i.e., in the presence of nucleotides and an agent for polymerization such as DNA polymerase and at a suitable temperature and pH.
  • the primer is preferably single stranded for maximum efficiency in amplification, but may alternatively be double stranded.
  • the primer is first treated to separate its strands before being used to prepare extension products.
  • the primer is an oligodeoxyribonucleotide.
  • the primer must be sufficiently long to prime the synthesis of extension products in the presence of the agent for polymerization.
  • the exact lengths of the primers will depend on many factors according to the particular application, including temperature and source of primer.
  • the oligonucleotide primer typically contains at least 15, more preferably 20 or more nucleotides, which are identical or complementary to the template and optionally a tail of variable length which need not match the template. The length of the tail should not be so long that it interferes with the recognition of the template. Short primer molecules generally require cooler temperatures to form sufficiently stable hybrid complexes with the template.
  • the primers herein are selected to be “substantially" complementary to the different strands of each specific sequence to be amplified. This means that the primers must be sufficiently complementary to hybridize with their respective strands. Therefore, the primer sequence need not reflect the exact sequence of the template. For example, a non- complementary nucleotide fragment may be attached to the 5' end of the primer, with the remainder of the primer sequence being complementary to the strand. Alternatively, non- complementary bases or longer sequences can be interspersed into the primer, provided that the primer sequence has sufficient complementarity with the sequence of the strand to be amplified to hybridize therewith and thereby form a template for synthesis of the extension product of the other primer.
  • Primer3 www-genome.wi.mit.edu/cgi-bin/primer/primer3.cgi
  • STSPipeline www- genome.wi.mit.edu/cgi-bin/www-STS_Pipeline
  • GeneUp for example, can be used to identify potential PCR primers.
  • Exemplary primers include primers that are 15 to 60 bases long, where at least between 70% of the bases are identical or complementary to at least 70% of the bases of a segment of the template sequence. In some examples described herein, primers that are 20 bases long are used.
  • primer hybridization sites refers to sections of a target nucleic acid to which a primer, i.e. a short oligonucleotide sequence ⁇ binds to.
  • the complementary binding of nucleic acid sequences is also known in the art as “hybridization”.
  • fusion refers to a hybrid construct, e.g. a gene or a protein, formed from two previously separate sources.
  • recombinant fusion proteins are created artificially by recombinant DNA technology for use in biological research or therapeutics.
  • the term “concatamer” refers to a long continuous DNA molecule that contains multiple copies of the same DNA sequences linked in series. These polymeric molecules are usually copies of an entire genome linked end to end and separated by cos sites (a protein binding nucleotide sequence that occurs once in each copy of the genome).
  • the technology disclosed herein enables linear amplification of chromatin immunoprecipitation (ChlP)-enriched DNA from very small amounts of sample or starting material.
  • the technique disclosed herein further combines linear amplification with in vitro transcription and subsequently, a library preparation protocol to generate sequencing ready libraries from as low as 50pg of starting material and lower.
  • the described method combines linear DNA amplification and library preparation in a single protocol and concurrently allows for the generation of readily sequenceable libraries from low starting concentrations of genetic material.
  • the addition of a T7 promoter primer is incorporated in the standard library preparation protocol.
  • the present invention refers to a kit for linear amplification of a DNA molecule according to the method as disclosed herein.
  • the present invention refers to the amplification of nucleic acid sequences from samples containing low concentrations of a target nucleic acid. While there are methods known in the art that may be used for the amplification of nucleic acid sequences, such as polymerase chain reaction, these exponential amplification methods also have their disadvantages, one of the most prominent ones being the inability to initiate stable amplification in samples with low template nucleic acid concentration, e.g. concentrations that are lower than the nanogram (ng) range.
  • ng nanogram
  • the starting amount of nucleic acid is between lOpg to lOOng, between lOpg to 50pg, between 50pg to lOOpg, between lOpg to 20pg, between 20pg to 30pg, between 30pg to 40pg, between 40pg to 50pg, between 50pg to 60pg, between 60pg to 70pg, between 70pg to 80pg, between 80pg to 90pg, between 90pg to lOOpg, between lOOpg to 200pg, between 200pg to 300pg, between 300pg to 400pg, between 400pg to 500pg, between 500pg to lOOOpg, between lng to lOng, between lOng to 50ng, between 50ng to lOOng, about l Opg, about 25pg, about 75pg, about 125pg, about 250pg, about 350pg, about 450pg, about 550pg, about
  • the term "exponential amplification” refers to a method of nucleic acid amplification wherein a specific template undergoes multiple rounds of primer-depleting amplification, resulting in an exponential increase in the number of copies made of the template with each round.
  • linear amplification refers to another method of nucleic acid amplification, whereby the number of copies made of a specific template increases in a linear fashion. The methodical differences between the exponential and linear amplification is the choice of polymerase, as well as the type of primers used.
  • An exponential amplification only requires primers that are capable of binding (annealing) to the target nucleic acid fragment as the polymerase, for example the Thermus aquaticus (Taq) polymerase, is capable of binding to the resulting double-stranded nucleic acid section and initiating nucleic acid polymerisation from there. No preparation of the target DNA is required.
  • a linear amplification requires the use of specially designed primers that includes a promoter site for efficient initiation by the utilized polymerase. Furthermore, the 3' ends of the target nucleic acid sequences have to be modified in order for these primers to anneal to the target nucleic acid sequence.
  • the concentration of primers required in an exponential amplification is much higher than the concentration of primers required in a linear amplification.
  • exponential amplification of nucleic acids requires temperature cycling, i.e. the use of different temperatures at each stage of the process, i.e. for denaturing the nucleic acid template, annealing the requisite primers and extending said primers.
  • a linear amplification is performed at a single, constant temperature.
  • nucleic acid library refers to a collection of nucleic acid fragments representing the transcriptome of a specific subject. These nucleic acid fragments are stored in host cells and can be amplified, and thus extracted, from the nucleic acid library using amplification methods known in the art. These nucleic acid libraries usually consist of cDNA sequences, but may also include or be made exclusively of genomic DNA.
  • the method as disclosed herein, for the sake of understanding, may be divided into various steps, which are undertaken to enable linear amplification with concurrent library enrichment and may be summarized as follows: end repair, tailing, adapter ligation, in vitro transcription/reverse transcription and amplification and/or multiplexing.
  • the present invention refers to a method of linear amplification of a DNA molecule, comprising the steps of (a) isolating a double-stranded DNA molecule from a DNA sample comprising the double-stranded DNA molecule to be amplified, (b) ligating a DNA adapter to a single nucleotide overhang at the tail of the DNA molecule obtained in the previous step, and (c) linear amplification of the modified DNA molecule obtained in the previous step to form a RNA amplification product.
  • the DNA molecule is a double-stranded DNA molecule.
  • Samples that can be used for the purpose of the invention disclosed herein include, but are not limited to, cell culture samples, cell lysate, blood, serum, urine, cells, organs, tissues, bone, bone marrow, lymph, lymph nodes, endothelial cells, vascular tissue, and skin.
  • Samples including genetic material generated by methods such as, but not limited to, RNA- Sequencing, chromatin isolation by RNA purification-sequencing (ChlRP-Seq), assay for transposase-accessible chromatin using sequencing (ATAC-Seq), genomic paired-end tags (gPET), chromatin interaction analysis by paired-end tag sequencing (ChlA-PET), exome- sequencing and whole genome sequencing, can also be used for the purpose of the invention disclosed herein.
  • methods such as, but not limited to, RNA- Sequencing, chromatin isolation by RNA purification-sequencing (ChlRP-Seq), assay for transposase-accessible chromatin using sequencing (ATAC-Seq), genomic paired-end tags (gPET), chromatin interaction analysis by paired-end tag sequencing (ChlA-PET), exome- sequencing and whole genome sequencing, can also be used for the purpose of the invention disclosed herein.
  • exome refers to the part of the genome formed by exons, i.e. the sequences which when transcribed remain within the mature RNA after introns are removed by RNA splicing.
  • An exome consists of all DNA that is transcribed into mature RNA in cells of any type and is distinct from the transcriptome, which is the RNA that has been transcribed only in a specific cell population.
  • the isolation of target DNA, or any other nucleic acid can be performed by methods known in the art, which include, but are not limited to, mechanical shearing, enzymatic isolation, phenol-chloroform extraction, polyethylene glycol (PEG) precipitation, centrifugation, density gradient centrifugation, spin column purification methods, anion exchange chromatography, magnetic beads and solid phase extraction.
  • methods known in the art include, but are not limited to, mechanical shearing, enzymatic isolation, phenol-chloroform extraction, polyethylene glycol (PEG) precipitation, centrifugation, density gradient centrifugation, spin column purification methods, anion exchange chromatography, magnetic beads and solid phase extraction.
  • nucleic acid purification may be required between downstream amplification and isolation steps.
  • Methods with which nucleic acid purification can be performed include, but are not limited to, alcohol/ethanol precipitation, the Boom method, phenol-chloroform extraction, as well as bead immobilization and spin column purification methods.
  • Beads suitable for such purposes include, but are not limited to, magnetic beads, ligand/affinity beads, nickel-agarose beads, highly porous polymer beads, polystyrene beads, sepharose beads, calmodulin beads and solid phase reversible immobilization (SPRI) beads.
  • SPRI beads are magnetic particles coated with carboxyl groups (in the form of succinic acid) that can bind DNA non- specifically and reversibly. If added to the DNA in the presence of polyethylene glycol (PEG) and salt (usually NaCl), they are an alternative to gel extraction with standardized and quick, binding and elution steps.
  • PEG polyethylene glycol
  • salt usually NaCl
  • the method as described herein further comprises using solid phase reversible immobilization beads to purify DNA between steps.
  • ligases As used herein, the terms “ligases”, “ligation enzymes” or “synthetases”, are enzymes that can catalyse the joining of two nucleic acids fragments (i.e. the act of "ligation” or “ligating”).
  • a DNA ligase is a specific type of enzyme that facilitates the joining of DNA strands together by catalysing the formation of a phosphodiester bond.
  • Ligases play a role in repairing single-strand breaks in double-stranded DNA in living organisms, but some forms (e.g. the DNA ligase IV) may specifically repair double-strand breaks, i.e. a break in both complementary strands of DNA.
  • Ligases can be chosen according to various experimental parameters and include, but are not limited to, T3 DNA ligase, T4 DNA ligase, T7 DNA ligase, Taq ligase and E. coli ligase. [0035] .
  • the method is as described herein, wherein step (b) is in the presence of a composition comprising at least one enzyme, wherein the at least one enzyme comprises a DNA ligase.
  • the DNA ligase is a T4 DNA ligase.
  • the method as descried herein further comprises the step of modifying the isolated double-stranded DNA molecule of step (a) as previously disclosed to generate a blunt-ended (end-repaired) DNA molecule.
  • nucleic acid fragments that have heterogeneous ends with 5' or 3' overhangs of varying lengths.
  • Some downstream applications require these ends to be treated to produce blunt ends, i.e. both strands of a nucleic acid molecule terminate in a base pair, in order to be effective substrates, e.g. for further ligation reactions. This process is also known as (DNA) blunting or end repair.
  • DNA DNA blunting or end repair.
  • Various enzymatic treatments known in the art can be used to generate blunt ends.
  • Single-strand specific exonucleases for example mung bean nuclease, can be used to digest the single stranded regions, leaving the nucleic acid fragments with blunt ends.
  • Other types of nucleic acid molecule ends known in the art are overhangs, frayed ends and cohesive or sticky ends, and may also be generated according to the downstream application requirement.
  • exonuclease or “exonuclease activity”, as used herein, refers to enzymes that cleave nucleotides one at a time from the end (exo) of a polynucleotide chain. Exonulceases are closely related to the endonucleases, which cleave phosphodiester bonds in the middle (endo) of a polynucleotide chain.
  • blunt ends can also be produced by T4 DNA polymerase, which has a single-strand exonuclease that removes 3' overhangs and a DNA polymerase activity that fills in 5' overhangs.
  • the T4 DNA polymerase is augmented by the Klenow fragment of E. coli DNA polymerase I.
  • Such enzymes known to be capable of such single-strand exonuclease activity also known as blunt-end restriction exonucleases, include, but are not limited to, T4 DNA polymerase, Klenow fragment (3' ⁇ 5' exo " ), mung bean nuclease, DNA Polymerase I - large (Klenow) fragment and Exonuclease I.
  • the method is as described herein, wherein the at least one enzyme has a 3' ⁇ 5' exonuclease activity and/or 5' ⁇ 3' polymerase activity.
  • the at least one enzyme is selected from the group comprising or consisting of a T4 DNA polymerase, a Bacillus stearothermophilus (Bst) polymerase, an E. coli polymerase I and a lenow fragment of E. coli DNA polymerase I or a combination thereof.
  • RNA polymerases (type I to III) catalyse the synthesis of RNA using a template that is either an existing DNA strand (DNA-dependent RNA polymerase) or an RNA strand.
  • Type I is responsible for the synthesis of ribosomal RNA, type II for messenger RNA (mRNA) synthesis and type III for transfer RNA (tRNA) synthesis.
  • mRNA messenger RNA
  • tRNA transfer RNA
  • DNA polymerases catalyse the elongation of a new DNA strand during DNA replication, using an existing DNA strand as a template.
  • RNA-directed DNA synthesis is more commonly known as reverse transcriptase.
  • Klenow fragment refers to a large protein fragment that is produced when DNA polymerase I from E. coli is enzymatically cleaved by the protease subtilisin. The resulting Klenow fragment thus retains its 5' ⁇ 3' polymerase activity and the 3' ⁇ 5' exonuclease activity for removal of pre-coding nucleotides and proofreading, but loses its 5' ⁇ 3' exonuclease activity.
  • the generation of the blunt-ended DNA molecule is in the presence of a composition comprising dNTP and at least one enzyme.
  • dNTP deoxyribonucleo tides triphosphate molecule, which is the monomer, or single unit, of DNA, or deoxyribonucleic acid.
  • deoxyribonucleotides triphosphate molecules There are four different types of deoxyribonucleotides triphosphate molecules known in the art: deoxyadenosine triphosphate (dATP), deoxycytidine triphosphate (dCTP), deoxyguanosine triphosphate (dGTP) and (deoxy-) thymidine triphosphate (dTTP).
  • dATP deoxyadenosine triphosphate
  • dCTP deoxycytidine triphosphate
  • dGTP deoxyguanosine triphosphate
  • dTTP deoxy-) thymidine triphosphate
  • Artificial deoxyribonucleotides triphosphate molecules are also known, as well as rare, tautomeric forms of deoxyribonucleotides triphosphate molecules.
  • blunt ended nucleic acid fragments In order to facilitate the further reactions, the blunt ended nucleic acid fragments often require phosphorylation.
  • blunt end cloning reactions are known to be less efficient compared to sticky end ligations due to the absence of the hydrogen bonding inherent to sticky ends. This absence means that the interactions between the vector and insert ends is fleeting, so a successful ligation relies on a transient association between 5' phosphate and 3'hydroxyl groups, caught at precisely the right time by the ligase.
  • blunt ends require phosphorylation. Enzymes that may be used for this purpose include, but are not limited to phosphorylases, phosphotransferases and kinases.
  • kinases that may be used for this purpose include, but are not limited to, T4 polynucleotide kinase, nucleoside-phosphate kinase and nucleoside monophosphate (NMP) kinases.
  • the method as described herein further comprises a polynucleotide kinase, wherein the polynucleotide kinase is capable of phosphorylating the 5 'end of the blunt-ended DNA molecule.
  • the polynucleotide kinase is a T4 polynucleotide kinase.
  • Tailing is a method by which one or more nucleotides may be added to the end of single or double stranded nucleic acids, resulting in the incorporation of one or more non- template nucleotides.
  • PCR products generated using a non-proofreading DNA polymerase, such as Tag DNA polymerase which lacks 3 ' ⁇ 5 ' exonuclease activity, have a single template-independent nucleotide at the 3 ' end of each DNA strand.
  • This single-nucleotide overhang which can be an A (adenosine) residue, allows hybridization with and cloning into vectors, for example T vectors, which are vectors named for their complementary 3 ' single T (thymidine) overhang.
  • PCR products generated using a proofreading DNA polymerase, such as Pfu DNA polymerase have blunt ends and must be cloned into a blunt-ended vector or need a single 3 ⁇ overhang added to ligate into a vector.
  • a typical DNA tailing reaction can incorporate anywhere from 1 to 3, 1 to 5, 1 to 10, 5 to 10, 10 to 20, 10 to 50, 25 to 50, 50 to 100, 100 to 200, 200 to 300, 75 to 125 or 150 to 250 nucleotides, depending on the reaction conditions selected and the type of nucleotides added to the reaction.
  • Tailing usually occurs at the 3 ' end of a blunt DNA fragment, thus preventing concatamer formation during subsequent ligation steps.
  • tailed DNA may be ligated to adaptors or cloning vectors with complementary overhangs, e.g. dT in case of a dA tail.
  • An A (adenosine) residue can be added by incubating the PCR fragment with dATP and a non-proofreading DNA polymerase, which will result in the addition of a single 3' A residue.
  • T (thymidine) residue would be achieved by incubation of a nucleic acid fragment with dTTP and the appropriate enzyme. Having such a single nucleotide overhang at the end of nucleic acid fragments facilitates downstream applications using said nucleic acid fragments.
  • the later ligation of an adaptor sequence to such a single nucleotide overhang makes the resulting nucleic acid fragment amenable for direct sequencing, as it is known in the art that repeating single nucleic acids or whole nucleic acid sequences are difficult to sequence.
  • Blunt ended restriction enzyme digested nucleic acid fragments may also be tailed using this method.
  • Enzymes capable of catalysing the addition of nucleotides to the 3' end of a nucleic acid molecule are also known as terminal deoxynucleotidyltransferases (TdTases).
  • These enzymes include, but are not limited to, terminal transferases, VentR (exo ) DNA polymerases, Bst DNA polymerase exo " , a Thermus aquaticus (Taq) polymerase and Klenow fragment of E. coli DNA polymerase I (3 ' ⁇ 5 ' Exo " ).
  • the method as described herein further comprises reacting the blunt-ended DNA molecule with single type dNTPs to obtain DNA molecules with a single nucleotide overhang at the 3 '-ends.
  • the method is as described herein, wherein the single nucleotide overhang is an adenosine.
  • the overhang is a guanine.
  • the method as described herein further comprises adding a composition comprising at least one enzyme capable of adding a single nucleotide tail to a blunt-ended DNA.
  • the at least one enzyme is selected from the group comprising or consisting of a Klenow fragment of E. coli DNA polymerase I, VentR (exo ) DNA Polymerase, Bst DNA polymerase exo- and a Thermus aquaticus (Taq) polymerase.
  • the present invention also provides for the addition of further sequences to the 5' or 3' ends of the generated nucleic acid fragments.
  • the method as described herein further comprises adding a RNA polymerase promoter nucleotide sequence to the DNA molecule obtained under step (b) as previously described.
  • adapter refers to the addition of a short, chemically synthesized, double stranded DNA molecule which is used to link the ends of two other DNA molecules.
  • the addition of an adapter to a target DNA molecule may be used, for example, to add sticky ends to cDNA allowing it to be ligated into the plasmid much more efficiently.
  • Adapters may also be used to link the ends of two DNA molecules that have different sequences at their ends.
  • a conversion adapter may be used to join a DNA sequence cleaved with one restriction enzyme, for example Eco Rl, with a vector that had been cleaved with another enzyme, Bam HI .
  • this adapter can be used to convert the cohesive end produced by Bam HI to one produced by Eco Rl or vice versa.
  • the length of the adapter is chosen according to the required function and intended purpose.
  • Adapters usually consist of a core sequence and an enzyme-specific sequence. The enzyme- specific sequence allows the ligation of the adapters to the resulting restriction fragments without restoring the original restriction sites. In this way, ligated adapters create a target site for primers in the subsequent amplification reactions. A person skilled in the art would be able to ascertain the sequence of the adapter required from the intended downstream application.
  • genetic adapters have a length of between 10 to 50 base pairs, between 10 to 20, between 20 to 30, between 30 to 40, between 40 to 50, between 50 to 60, between 60 to 79, between 25 to 45 base pairs, about 15 base pairs, about 25 base pairs, about 35 base pairs, about 45 base pairs, about 55 base pairs and about 65 base pairs.
  • the genetic adapter has a length of 62 base pairs.
  • the D A adapter of step (b) comprises a first single-stranded oligonucleotide and a second single-stranded oligonucleotide, wherein the first single- stranded oligonucleotide and the second single-stranded oligonucleotide comprise (aa) a first region capable of forming a double-stranded oligonucleotide by annealing of the first oligonucleotide to the second oligonucleotide based on complementary binding; (bb) a second region in the first oligonucleotide that comprises a nucleotide sequence non- complementary to the sequence of the second oligonucleotide and wherein the second region is capable of hybridizing with a sequencing primer; and (cc) optionally a third region in the second oligonucleotide, wherein the third region comprises (iv) a nucleotide sequence capable of hybrid
  • forked adapter refers to DNA adapters which have a "Y” configuration.
  • Standard libraries for example for Illumina sequencing, are prepared by ligating a single "Y” or “forked” adapter to both ends of nucleic fragments.
  • the DNA adapter is a forked adapter wherein the first single-stranded oligonucleotide comprises or includes or consists of the nucleotide sequence of SEQ ID NO: 1 (A2 : ACACTCTTTCCCTACACGACGCTCTTCCGATC*T) and the second single-stranded oligonucleotide comprises or includes or consists of the nucleotide sequence of SEQ ID NO: 2 (Al : GATCGGAAGAGCACACGTCT).
  • These adapters made by annealing oligonucleotides with both complementary and non-complementary sequences, have, at one end, a region of double stranded DNA that is required for a ligase, for example a T4 DNA ligase, to join adapters to nucleic acid sequence.
  • the other end of the adapter is comprised of single stranded, divergent sequences that serve as binding sites for a pair of primers that, for example using PCR, generate nucleic acid fragments with different adapter sequences on each end.
  • the method is as described herein, wherein the first single-stranded oligonucleotide sequence comprises a 3 '-end T-tail overhang and wherein the second region comprises at least a region complementary to sequencing primer hybridization sites.
  • the method as described herein further comprises adding a RNA polymerase promoter nucleotide sequence to the DNA molecules as obtained previously.
  • the method as described herein further comprises, after step (iv) as described herein, (x) annealing at least one primer comprising a first region having a nucleotide sequence comprising the RNA polymerase promoter sequence and a second region comprising or consisting of nucleotides complementary to the 3 'end of said DNA molecule; and (y) extending the annealed product of step (x) in the presence of a polymerase and dNTPs.
  • the at least one primer is a promoter-adapter fusion primer comprising a promoter for a RNA polymerase and is capable of annealing to the second single-stranded oligonucleotide of the DNA adapter as previously described based on complementary binding.
  • the adapter sequence is fused to the T7 promoter sequence, resulting in a fusion primer.
  • the at least one primer has a nucleotide sequence of SEQ ID NO. 3 (5 ' - A ATTAAT ACGACTC ACT AT AGGG AG ACGTGTGCTCTTCCG ATC-3 ' ) .
  • the method as previously described further comprises a second primer, wherein the second primer is capable of annealing to the reverse complement of the first single-stranded oligonucleotide of the DNA adapter as described herein based on complementary binding.
  • the second primer comprises the nucleotide sequence of SEQ ID NO. 4 (5' ACACTCTTTCCCTACACGACGCTCTTCCGATC-3').
  • the second primer is a promoter adapter fusion primer comprising a promoter for a RNA polymerase.
  • the second primer has a nucleotide sequence of SEQ ID NO. 5 (5'- AATTAATACGACTCACTATAGGGACACTCTTTCCCTACACGACGCTCTTCCGATC- 3').
  • the promoter sequences added to the primer sequence may be changed accordingly.
  • the at least one primer comprises a T7 RNA polymerase promoter sequence, SP6 RNA polymerase promoter sequence, or a T3 RNA polymerase promoter sequence.
  • the polymerase is selected from the group consisting of a Klenow DNA polymerase, an E. coli DNA polymerase I, or any thermophilic DNA polymerase select from the group consisting of Phusion (Pyrococcus furiosus), Vent (Thermus aquaticus), Pfx (Thermococcus sp. Strain KOD), Pfu (Pyrococcus furiosus) and Q5 (recombinant high-fidelity DNA polymerase).
  • the present invention describes the amplification of the modified nucleic acid fragments. This is done using a method known as in vitro transcription. This method is a linear nucleic acid amplification method, as opposed to the generally known exponential amplification method defined previously. The linear amplification is then coupled with reverse transcription, which results in the generation of a complementary DNA (cDNA) from the RNA generated via in vitro transcription.
  • cDNA complementary DNA
  • the method as described herein further comprises after step (c) as defined herein, the step of (i) reverse transcribing of the RNA amplification product to generate single-stranded DNA; (ii) synthesizing a second strand of the single-stranded DNA to obtain double-stranded DNA fragments; and (iii) optionally, repeating steps (c) as described herein and (i)-(ii) at least once.
  • reverse transcription refers to the process of synthesizing DNA using RNA as a template and reverse transcriptase as the catalytic enzyme.
  • the term “reverse” thus implies that this process, the transcription, usually occurs in the other direction, i.e. the generation of RNA from DNA.
  • a reverse transcriptase is needed, for example, for the replication of retroviruses (e.g., HIV), and reverse transcriptase inhibitors are widely used as antiretroviral drugs.
  • Reverse transcriptase activities are also associated with the replication of chromosome ends (e.g. telomerase) and some mobile genetic elements (e.g. retrotransposons).
  • the method is as described herein, wherein step (i) further comprises adding to the sample a reverse transcriptase (RT) and a pair of primers.
  • the reverse transcription is selected from the group consisting of a modified Moloney Murine Leukemia Virus (M-MLV), a Moloney Murine Leukemia Virus (M-MuLV) RT, and an Avian Myeloblastosis Virus (AMV) RT.
  • the reverse transcriptase is a modified M-MLV reverse transcriptase (modified Moloney Murine Leukemia Virus) with reduced RNase H activity.
  • Other enzymes known to be useful in reverse transcription may be, but are not limited to, reverse transcriptases isolate from viruses, such as human T-cell lymphotrophic virus I (HTLV-I) and Telomerase reverse transcriptase.
  • step (ii) further comprises adding to the sample a reverse transcriptase, a pair of primers and a DNA polymerase to the single- stranded DNA of step (ii).
  • steps (i) and (ii) are performed in a single reaction vessel by adding to the RNA amplification product, a reverse transcriptase, a thermostable DNA polymerase, a pair of primers and dNTPs for first and second strand synthesis using the RNA amplification product as a template.
  • the second strand synthesis in step (ii) is carried out using a thermostable Taq DNA polymerase composition.
  • the composition further comprises a recombinant Taq DNA polymerase, Pyrococcus species GB-D polymerase, and Taq antibodies, which inhibit polymerase activity at about 15°C to about 37°C and a proof reading polymerase such as Pfu (Pyrococcus furiosus) DNA polymerase, Pfx (KOD polymerase), Phusion (Pyrococcus sp.), or Q5 (recombinant high fidelity polymerase).
  • a proof reading polymerase such as Pfu (Pyrococcus furiosus) DNA polymerase, Pfx (KOD polymerase), Phusion (Pyrococcus sp.), or Q5 (recombinant high fidelity polymerase).
  • second strand synthesis refers to the synthesis of the complementary DNA strand from an existing single-stranded DNA or DNA-RNA hybrid.
  • a DNA-RNA hybrid is the template, as for example when the product of a reverse transcription reaction is used as template, the RNA removal by (enzymatic) digestion is required prior to second strand synthesis.
  • an RNAse such as RNAseH may be used to nick the DNA/RNA hybrid, and a DNA polymerase used to catalyse the second strand cDNA synthesis using the RNA fragments as primers.
  • RNAseH refers to ribonuclease H, an endoribonuclease that specifically degrades the RNA strand in RNA-DNA hybrids.
  • this second strand synthesis produces a second strand DNA. copy that lacks the last 5-20 base pairs at the 5' termini.
  • the reaction may then be treated with DNA ligase to ligate all of the pieces of DNA that make up the second strand, since second strand synthesis starts at multiple locations from random RNA primers left following RNaseH treatment.
  • T4 DNA polymerase, Pfu polymerase or other polymerases having 3 ⁇ 5' exonuclease activity may be added to polish or blunt the 3' end of the first strand.
  • the primers containing an RNA polymerase binding site and a tail are concurrently present during the second strand synthesis step, abolishing the requirement of a polishing step.
  • Enzymes that can be used for second strand synthesis include, but are not limited to, E. coli Polymerase I, Taq polymerase, lenow, T7, T4 DNA polymerase, phusion and pfu polymerases.
  • the resulting complementary DNA has a base sequence that is complementary to that of a messenger RNA (mRNA) sequence. That means that unlike genomic DNA, complementary DNA contains no intron (i.e. non-coding) sequences.
  • Uses for complementary DNA are varied and numerous, the most common uses being gene cloning or as gene probes or in the creation of a cDNA library.
  • Complementary DNA libraries are commonly used when reproducing eukaryotic genomes, as the amount of information is reduced to remove the large numbers of non-coding regions from the library.
  • Complementary DNA libraries are used to express eukaryotic genes in prokaryotes.
  • Prokaryotes do not have introns in their DNA and therefore do not possess enzymes are capable of excising the same during transcription process. However, cDNA does not have introns and therefore can be expressed in prokaryotic cells. Complementary DNA libraries are most useful in reverse genetics where the additional genomic information is of less use. Also, it is useful for subsequently isolating the gene that codes for specific messenger RNA (mRNA).
  • mRNA messenger RNA
  • the method as previously defined further comprises sequencing of the amplified DNA fragment, optionally by high-throughput sequencing.
  • high-throughput sequencing refers to a particular variation of nucleic acid sequencing, whereby the term “high-throughput” refers to the analysis of a large number of sequences within a short period of time.
  • Nucleic acid sequencing refers to the process of elucidating the nucleotide sequence of, for example, a DNA fragment.
  • One method used for sequence elucidation is the Sanger method, also known as the dideoxy or chain-termination method.
  • nucleic acid sequencing examples include, but are not limited to, maxam-gilbert sequencing, shot gun sequencing, de novo sequencing and next-generation sequencing methods, which include, but are not limited to, genome sequencing, genome re-sequencing, transcriptome profiling (RNA-Sequencing), DNA-protein interactions (ChlP-sequencing), sequencing by ligation (SOLiD sequencing), sequencing by synthesis, pyrosequencing, ion semiconductor (Ion Torrent sequencing), single-molecule real-time sequencing, massively parallel signature sequencing (MPSS), Polony sequencing, DNA nanoball sequencing, sequencing by hybridization and sequencing with mass spectrometry.
  • RNA-Sequencing transcriptome profiling
  • ChlP-sequencing DNA-protein interactions
  • SOLiD sequencing sequencing by ligation
  • sequencing by synthesis pyrosequencing
  • Ion semiconductor sequencing ion semiconductor sequencing
  • MPSS massively parallel signature sequencing
  • Polony sequencing DNA nanoball sequencing, sequencing by hybridization and sequencing with mass spectrometry.
  • the DNA sample is obtained from chromatin immunoprecipitation (ChIP), sequential chromatin immunoprecipitation (reChIP), genomic paired-end tags (gPET), chromatin interaction analysis by paired-end tag sequencing (ChlA- PET), chromosome conformation capture (3C), genome conformation capture related chromosome conformation capture (Hi-C), exome capture or double stranded cDNA for RNA-Sequencing.
  • DNA polymerases and terminal transferases require a divalent cation for catalytic activity.
  • the preferred divalent cation is Mg 2+ , although other cations, such as Mn 2+ or Co 2+ can activate DNA polymerases.
  • Co 2+ is preferred, although Mg 2+ and Mn 2+ can also be used.
  • Mn is preferred as the divalent cation.
  • the divalent cation is typically included as a salt, for example a chloride, acetate or sulphate salt, e.g.
  • usable cation concentrations in a Tris buffer will be in a range from 0.5 to 7 mM or between 0.5 and 2 mM.
  • usable divalent cation concentrations in a Tris buffer will be in a range from 0.5 to 10 mM MgCl 2 .
  • buffer refers to a solution containing a buffering agent or a mixture of buffering agents and, optionally, a divalent cation and a monovalent cation.
  • a buffer solution may also contain a reducing agent, such as dithiothreitol or mercaptoethanol.
  • reaction mixture refers to an aqueous solution comprising the various reagents used for a given enzymatic reaction. These may include enzymes, aqueous buffers, salts, amplification primers, target nucleic acid, and nucleoside triphosphates (NTPs) or deoxyribonucleoside triphosphates (dNTPs).
  • NTPs nucleoside triphosphates
  • dNTPs deoxyribonucleoside triphosphates
  • the mixture can be either a complete or incomplete amplification reaction mixture.
  • the mixture may contain all the buffering elements required for enzymatic activity, but lack certain enzymes or dNTPs.
  • heat treatment may be used to denature enzymes in a sample, thus stopping the reaction. Heat treatment should be sufficient to denature the enzyme in the sample. The degree and duration of the treatment can be easily determined by the skilled person, as the denaturation temperature of commercially available enzymes is known.
  • the heat treatment is performed at a temperature that does not denature the DNA in the sample. This is particularly important at the stage of using a 5'-3' DNA polymerase to synthesise DNA complementary to the primer overhangs, as it is the lack of denaturation of the strands before end filling which permits the creation of double- stranded DNA fragments with an RNA polymerase promoter site at both ends.
  • Suitable heat treatment may comprise heating to between 65°C and 75°C, for example to 65°C , 66°C , 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C or 75°C for a period of at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 60 minutes or more, or overnight.
  • heat treatment may comprise heating the sample to 72 °C for 10 minutes.
  • Incubation of a sample with enzyme involves maintaining the sample at a temperature compatible with enzyme activity for an appropriate period. Incubation temperatures for most enzymes are between 20°C and 47 °C, depending on the source organism of the enzyme, for example 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41 °C, 42°C, 43°C, 44°C, 45°C, 46°C or 47°C.
  • incubation is at or around 37°C, though certain enzymes such as reverse transcriptases function most efficiently at a higher temperature, preferably at or around 42°C. Incubation may be carried out for example for 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 60 minutes or more, or overnight. Certain enzymes, such as terminal transferase, should be incubated for shorter periods, for example 20 minutes. The optimal temperature and period of incubation can be readily determined by the skilled person based on the known properties of these enzymes.
  • the method is as previously described, wherein the method is carried out in a same container.
  • all of the steps may be carried out in a single container.
  • all of the steps up to and including the reverse transcriptase step may be carried out in the same container.
  • This has the advantage that the steps are carried out in the same container, for example a single reaction tube or microtitre plate, without the need to transfer the sample between containers or apply it to columns, all of which the processes risk losing the isolated nucleic acids.
  • These steps may also be automated, which is of particular use in large scale or high-throughput analysis, for example large scale analyses of patients to determine their epigenetics or genomic profiles.
  • a genetic marker includes a plurality of genetic markers, including mixtures and combinations thereof.
  • the term "about”, in the context of concentrations of components of the formulations, typically means +/- 5% of the stated value, more typically +/- 4% of the stated value, more typically +/- 3% of the stated value, more typically, +/- 2% of the stated value, even more typically +/- 1% of the stated value, and even more typically +/- 0.5% of the stated value.
  • range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosed ranges. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.
  • chromatin immunoprecipitation (ChlP)-enriched double stranded DNA was blunt ended by Klenow and T4 DNA polymerase. This blunt ended DNA was treated with Taq polymerase to add adenosine ("A") overhangs to the 3' end. This enabled efficient ligation of the forked Illumina multiplexing adaptors to the blunt ended DNA. All reactions were carried out on Ampure beads to prevent sample loss.
  • the ligated DNA was then eluted off the Ampure beads and a T7 promoter sequence was added by annealing a novel T7-adaptor fusion primer, and extending the annealed product by application of a Klenow polymerase.
  • the double-stranded DNA product which now has a T7 promoter sequence at one end, was subjected to T7 RNA polymerase- mediated in vitro transcription (IVT) at 37°C for 16 hours.
  • IVTT in vitro transcription
  • the in vitro transcription generated RNA was reverse transcribed and amplified via polymerase chain reaction in a single reaction tube using Illumina universal primer and multiplexing indexed primers.
  • the amplified library was then size-selected using Ampure beads.
  • Size selection was performed in a two-step fashion.
  • 0.65X Ampure beads were added to the in vitro transcription reaction mix, where "X" indicates the volume of the mix. After binding, the beads were retained on a magnet and the supernatant was transferred to a fresh tube. A volume of 0.15X of Ampure beads was added to the supernatant. After binding to the magnet, the target DNA was contained on Ampure beads. The supernatant was discarded. The Ampure beads were washed with 80% ethanol, dried and the target DNA eluted off the beads by the addition of lOmM Tris-HCl buffer (pH 8.0).
  • Linear amplified DNA is purified using a Qiagen RNeasy Mini Kit.
  • To prepare a 1.7 mL reaction tube add 50 iL of the IVT sample, 50 ⁇ ih RNase-free water, and 350 ⁇ L of the RLT Lysis Buffer into the reaction tube.
  • Add 250 of 100% ethanol and transfer rapidly into the kit provided microcentrifuge column. Centrifuge the columns at 12,000 rcf for 30 seconds. Discard supernatant from the collection tube. Wash column with 500 ⁇ iL RPE buffer, centrifuge at 12,000 rcf for 30 seconds and discard supernatant. Wash column again with 500 RPE buffer and centrifuge for a further 2 minutes.
  • RNA is bound to the membrane.
  • RT-PCR SSIII RT-PCR mix from Invitrogen

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Health & Medical Sciences (AREA)
  • Biophysics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Immunology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Analytical Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

La présente invention concerne un procédé d'amplification linéaire d'une molécule d'ADN, comprenant les étapes consistant à isoler une molécule d'ADN double brin à partir d'un échantillon d'ADN contenant la molécule d'ADN double brin devant être amplifiée; à ligaturer un adaptateur ADN à une saillie mononucléotidique située sur la queue de la molécule d'ADN obtenue précédemment; et à procéder à une amplification linéaire de la molécule d'ADN modifiée obtenue précédemment afin d'obtenir un produit d'amplification d'ARN. Dans des modes de réalisation préférés, les adaptateurs sont fourchus ou en forme de Y, et une amorce de fusion d'adaptateurs T7 est utilisée pour favoriser la transcription d'une polymérase T7. L'invention concerne également un procédé de génération de banques concurrentes à partir des mêmes molécules d'ADN.
PCT/SG2014/000463 2013-10-03 2014-10-03 Enrichissement de banques parallèles d'amplification Ceased WO2015050501A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
SG201307435 2013-10-03
SG201307435-6 2013-10-03

Publications (1)

Publication Number Publication Date
WO2015050501A1 true WO2015050501A1 (fr) 2015-04-09

Family

ID=52778983

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/SG2014/000463 Ceased WO2015050501A1 (fr) 2013-10-03 2014-10-03 Enrichissement de banques parallèles d'amplification

Country Status (1)

Country Link
WO (1) WO2015050501A1 (fr)

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105839196A (zh) * 2016-05-11 2016-08-10 北京百迈客生物科技有限公司 一种真核生物DNA的Hi-C高通量测序建库方法
WO2017100343A1 (fr) * 2015-12-07 2017-06-15 Arc Bio, Llc Procédés et compositions pour la fabrication et l'utilisation d'acides nucléiques de guidage
CN111378724A (zh) * 2018-12-27 2020-07-07 上海仁度生物科技有限公司 一种rna放大检测方法及检测试剂盒
CN111433359A (zh) * 2017-11-20 2020-07-17 Bioo科技公司 制备cDNA文库的方法
CN112680797A (zh) * 2021-02-04 2021-04-20 广州大学 一种去除高丰度rna的测序文库及其构建方法
WO2022046785A1 (fr) * 2020-08-28 2022-03-03 Applied Materials, Inc. Procédés de préparation de quantités importantes d'adn simple brin (adnsb)
WO2023227699A1 (fr) * 2022-05-25 2023-11-30 Epigenica Ab Ligature d'adaptateur

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008093098A2 (fr) * 2007-02-02 2008-08-07 Illumina Cambridge Limited Procedes pour indexer des echantillons et sequencer de multiples matrices nucleotidiques
WO2012150317A1 (fr) * 2011-05-05 2012-11-08 Institut National De La Sante Et De La Recherche Medicale (Inserm) Amplification d'adn linéaire

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008093098A2 (fr) * 2007-02-02 2008-08-07 Illumina Cambridge Limited Procedes pour indexer des echantillons et sequencer de multiples matrices nucleotidiques
WO2012150317A1 (fr) * 2011-05-05 2012-11-08 Institut National De La Sante Et De La Recherche Medicale (Inserm) Amplification d'adn linéaire

Non-Patent Citations (9)

* Cited by examiner, † Cited by third party
Title
"Illumunia Customer Sequence Letter", 11 October 2011 (2011-10-11), Retrieved from the Internet <URL:http://dnatech.genomecenter.ucdavis.edu/wp-content/uploads/2013/10/006.2011-10-11-Illumina-Customer-Sequence-Letter-upload-pdf> [retrieved on 20150206] *
"llumina Customer Sequence Letter", 8 July 2014 (2014-07-08), Retrieved from the Internet <URL:http://supportres.illumina.com/documents/documentation/chemistry_documentafion/ex periment-design/illumina-customer-sequence-letter.pdf> [retrieved on 20150206] *
BOWMAN, S.K. ET AL.: "Multiplexed Illumina sequencing from picogram quantities of DNA", BMC GENOMICS, vol. 14, no. 466, 2013, pages 1 - 7 *
HOEIJMAKERS, W.A.M. ET AL.: "Linear amplification for deep sequencing", NATURE PROTOCOLS, vol. 6, no. 7, 2011, pages 1026 - 1036 *
KOZAREWA, I. ET AL.: "Amplification-free Illumina sequencing-library preparation facilitates improved mapping and assembly of GC-biased genomes", NATURE METHODS, vol. 6, no. 4, 2009, pages 291 - 295 *
SCHMITT, M.W. ET AL.: "Detection of ultra-rare mutations by next-generation sequencing", PNAS, vol. 109, no. 36, 2012, pages 14508 - 14513 *
SENGUPTA, S. ET AL.: "Single Read and Paired End mRNA-Seq Illumina Libraries from 10 Nanograms Total RNA", JOURNAL OF VISUALIZED EXPERIMENTS, vol. 56, 2011, pages 3340 *
SHANKARANARAYANAN, P. ET AL.: "Single-tube linear DNA amplification (LinDA) for robust ChIP-seq", NATURE METHODS, vol. 8, no. 7, 2011, pages 565 - 567 *
VAN BAKEL, H. ET AL.: "Improved genome-wide localization by ChIP-chip using double-round T7 RNA polymerase-based amplification", NUCLEIC ACIDS RESEARCH, vol. 36, no. 4, 2008, pages E21 *

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10787662B2 (en) 2015-12-07 2020-09-29 Arc Bio, Llc Methods and compositions for the making and using of guide nucleic acids
WO2017100343A1 (fr) * 2015-12-07 2017-06-15 Arc Bio, Llc Procédés et compositions pour la fabrication et l'utilisation d'acides nucléiques de guidage
JP2018535689A (ja) * 2015-12-07 2018-12-06 アーク バイオ， エルエルシー ガイド核酸を作製および使用するための方法および組成物
JP6995751B2 (ja) 2015-12-07 2022-02-04 アーク バイオ， エルエルシー ガイド核酸を作製および使用するための方法および組成物
CN105839196B (zh) * 2016-05-11 2018-04-17 北京百迈客生物科技有限公司 一种真核生物DNA的Hi‑C高通量测序建库方法
CN105839196A (zh) * 2016-05-11 2016-08-10 北京百迈客生物科技有限公司 一种真核生物DNA的Hi-C高通量测序建库方法
CN111433359A (zh) * 2017-11-20 2020-07-17 Bioo科技公司 制备cDNA文库的方法
CN111378724A (zh) * 2018-12-27 2020-07-07 上海仁度生物科技有限公司 一种rna放大检测方法及检测试剂盒
CN111378724B (zh) * 2018-12-27 2024-03-22 上海仁度生物科技股份有限公司 一种rna放大检测方法及检测试剂盒
WO2022046785A1 (fr) * 2020-08-28 2022-03-03 Applied Materials, Inc. Procédés de préparation de quantités importantes d'adn simple brin (adnsb)
CN112680797A (zh) * 2021-02-04 2021-04-20 广州大学 一种去除高丰度rna的测序文库及其构建方法
CN112680797B (zh) * 2021-02-04 2023-09-26 广州大学 一种去除高丰度rna的测序文库及其构建方法
WO2023227699A1 (fr) * 2022-05-25 2023-11-30 Epigenica Ab Ligature d'adaptateur

Similar Documents

Publication Publication Date Title
US10876108B2 (en) Compositions and methods for targeted nucleic acid sequence enrichment and high efficiency library generation
EP3350732B1 (fr) Méthode de préparation d&#39;une bibliothèque de séquençage de nouvelle génération (ngs) à partir d&#39;un échantillon d&#39;acide ribonucléique (arn) et kit pour la mise en oeuvre de cette dernière
CA2929596C (fr) Compositions et procedes pour l&#39;identification d&#39;une lecture de sequencage en double
US20160130576A1 (en) Compositions and methods for directional nucleic acid amplification and sequencing
US12448648B2 (en) Controlled strand-displacement for paired end sequencing
WO2015050501A1 (fr) Enrichissement de banques parallèles d&#39;amplification
EP2585593A2 (fr) Procédés et compositions pour la production, l&#39;immortalisation d&#39;une banque de polynucléotides et l&#39;extraction de régions d&#39;intérêt
CN111542532B (zh) 酶法合成寡核苷酸的方法和体系
US11834657B2 (en) Methods for sample preparation
CN113811610A (zh) 用于改进cDNA合成的组合物和方法
EP4392577B1 (fr) Ensemble optimisé d&#39;oligonucléotides pour le codage à barres et le séquençage d&#39;arn en vrac
HK40057111A (zh) 生成双链衔接物的方法

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 14850724

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 14850724

Country of ref document: EP

Kind code of ref document: A1