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WO2020183188A1 - Procédés d'amplification d'acide nucléique - Google Patents

Procédés d'amplification d'acide nucléique Download PDF

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Publication number
WO2020183188A1
WO2020183188A1 PCT/GB2020/050635 GB2020050635W WO2020183188A1 WO 2020183188 A1 WO2020183188 A1 WO 2020183188A1 GB 2020050635 W GB2020050635 W GB 2020050635W WO 2020183188 A1 WO2020183188 A1 WO 2020183188A1
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Prior art keywords
adapter
oligonucleotide
adapter oligonucleotide
template dna
dna
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Gerard Brady
Alexandra CLIPSON
Dominic ROTHWELL
Caroline DIVE
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Cancer Research Technology Ltd
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Cancer Research Technology Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6865—Promoter-based amplification, e.g. nucleic acid sequence amplification [NASBA], self-sustained sequence replication [3SR] or transcription-based amplification system [TAS]
    • 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 improved, reliable and highly specific methods of amplifying DNA to produce an aRNA or DNA product, utilising novel adapter oligonucleotides, which can be used to identify genetic variations in biological samples.
  • NGS Next Generation Sequencing
  • circulating cell-free DNA has provided opportunities to analyse genetic materials from blood samples, without the risks typically associated with more invasive sampling methods.
  • tumour DNA circulating tumour DNA
  • the ctDNA may be analysed by sequencing cfDNA to detect sequence variants that are known to be specifically associated with various types of tumours or cfDNA copy numbers to detect the presence of tumours and their progression.
  • CTCs circulating tumour cells
  • a method for producing an aRNA product from template DNA comprising:
  • a secondary oligonucleotide comprising an RNA polymerase promoter sequence, wherein the sequence of the secondary oligonucleotide is complementary to the sequence of the adapter oligonucleotide;
  • the method of the present invention provides a number of advantages. Compared to other methods used to amplify template DNA the approach is simpler, more flexible and can provide increased sensitivity and specificity making it more suitable where input DNA is limited (for example in the analysis of cfDNA and single cells).
  • aRNA as used herein is used to refer to amplified RNA produced by an RNA polymerase from the template DNA of the present invention.
  • the method of the present invention comprises providing an adapter oligonucleotide comprising a RNA polymerase promoter sequence and transcribing template DNA by introducing a RNA polymerase.
  • RNA polymerase there are many RNA polymerases which could be utilised in the method of the present invention, such as T3 RNA polymerase, SP6 RNA polymerase and T7 RNA polymerase.
  • the RNA polymerase may comprise a T7 RNA polymerase and the RNA polymerase promoter sequence may comprise a T7 RNA polymerase promoter sequence.
  • the present inventors have shown that utilising a T7 RNA polymerase and associated promoter sequence results in a highly efficient, sensitive and specific method.
  • the RNA polymerase promoter sequence in the adapter oligonucleotide may comprise SEQ ID N0.1 or a fragment or variant thereof.
  • the T7 RNA polymerase promoter sequence is: ATTATGCTGAGTGATATCCC (SEQ ID NO. 1).
  • the RNA polymerase promoter sequence in the adapter oligonucleotide consists of SEQ I D NO.1.
  • the RNA polymerase promoter sequence in the secondary oligonucleotide may comprise SEQ ID NO.2 or a fragment or variant thereof.
  • the Reverse T7 RNA polymerase promoter sequence is: TAATACGACTCACTATAGGG (SEQ ID NO. 2).
  • the RNA polymerase promoter sequence in the secondary oligonucleotide consists of SEQ ID NO.2.
  • fragment or variant as used herein is used in its broadest sense to refer to a fragment or variant of the sequence disclosed which retains its function, i.e. in this case, the ability to act as an RNA polymerase promoter sequence thereby allowing an RNA polymerase to bind.
  • the fragment or variant comprises at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity with SEQ ID N0.1 or 2.
  • the fragment or variant has 100% sequence identity at positions 7, 8, 9, 10 and 11 of SEQ ID N0.1 , as shown underlined below:
  • the fragment or variant has 100% sequence identity at positions 7, 8, 9, 10 and 11 of SEQ ID NO:2, as shown underlined below: TAATACGACTCACTATAGGG
  • the sequence of the secondary oligonucleotide is complementary to the sequence of the adapter oligonucleotide.
  • This sequence complementarity may be by way of the RNA polymerase promoter sequences present in the adapter oligonucleotide and secondary oligonucleotide respectively.
  • the RNA polymerase promoter sequences in the adapter oligonucleotide and secondary oligonucleotide may be complementary or at least partially complementary in sequence.
  • the RNA polymerase promoter sequence in the adapter oligonucleotide comprises SEQ ID NO:1 or a fragment or variant thereof and the RNA polymerase promoter sequence in the secondary oligonucleotide comprises SEQ ID NO:2 or a fragment or variant thereof.
  • the secondary and adapter oligonucleotides may comprise further complementary sequences, in addition to the RNA polymerase promoter sequences.
  • Step ii) of the method of the present invention comprises ligating the 5’ end of the adapter oligonucleotide to the 3’ end of the template DNA to produce an adapter oligonucleotide- template DNA molecule.
  • the adapter oligonucleotide is configured so that a 5’ end of the adapter oligonucleotide can be ligated to a 3’ end of the template DNA.
  • the adapter oligonucleotide may be configured so that a 5’ end of the adapter oligonucleotide can be ligated to a 3’ end of the template DNA.
  • the adapter oligonucleotide may comprise a T-tail.
  • the template DNA may comprise an A-tail, facilitating adapter oligonucleotide and template DNA annealing (due to the corresponding A- and T-tails) such that they are in proximity for subsequent ligation.
  • the adapter oligonucleotide may comprise a 5’ sequence which is complementary to a 3’ sequence of the corresponding DNA template.
  • the DNA template may have been treated with a restriction enzyme resulting in an overhanging sequence.
  • the 5’ end of the adapter oligonucleotide may have a sequence corresponding to the overhanding sequence of the DNA template such that the adapter oligonucleotide and template DNA can anneal such that they are in proximity for subsequent ligation.
  • step ii) may comprise ligating the 5’ end of the adapter oligonucleotide to the 3’ end of the template DNA using a DNA ligase.
  • a DNA ligase suitable of ligating the 5’ end of the adapter oligonucleotide to the 3’ end of the template DNA may be used.
  • the DNA ligase may comprise a bacterial, bacteriophage (e.g. T4 DNA ligase) or mammalian DNA ligase (e.g. DNA ligase I, II, III or IV), for example.
  • the DNA ligase may comprise a T4 DNA ligase, for example KAPA-hyper DNA ligase, NEB Quick Ligation Kit, NEB T4 DNA Ligase, NEB Blunt/TA Ligase Master Mix or NEBNext® UltraTM II Ligation Module.
  • T4 DNA ligase for example KAPA-hyper DNA ligase, NEB Quick Ligation Kit, NEB T4 DNA Ligase, NEB Blunt/TA Ligase Master Mix or NEBNext® UltraTM II Ligation Module.
  • the DNA ligase may comprise KAPA- hyper DNA ligase.
  • the present inventors found that the use of KAPA-hyper DNA ligase is particularly effective in the method of the present invention (see for example, Figure 4).
  • the design of the adapter oligonucleotide allows ligation of the 5’ end of the adapter oligonucleotide to the 3’ end of the template DNA. This results in a contiguous molecule incorporating the RNA polymerase promoter at the 3’ end and the template DNA at the 5’ end. Due to the orientation of the adapter oligonucleotide-template DNA molecule there is no need to convert to a dsDNA molecule for the RNA polymerisation step since the ligated adapter oligonucleotide-template DNA molecule can be utilised as a template directly by the RNA polymerase.
  • ligation of the 5’ end of the adapter oligonucleotide to the 3’ end of the template DNA takes place between around 14 and 22°C, preferably between around 16 and 22°C, preferably at 20°C.
  • the method may further comprise dephosphorylating the template DNA, preferably prior to the ligation of step ii) of the present invention.
  • a 5’ phosphate of the template DNA is removed by treatment with a phosphatase enzyme.
  • any suitable phosphatase enzyme could be utilised, for example Shrimp Alkaline Phosphatase, Antarctic Phosphatase, Calf Intestinal Alkaline Phosphatase or FastAP Thermosensitive Alkaline Phosphatase.
  • the phosphatase comprises a heat labile phosphatase such as FastAP Thermosensitive Alkaline Phosphatase.
  • the present inventors have found that the removal of the 5’ phosphate from the template DNA reduces DNA self ligation and also reduces or blocks ligation of the 3’ end of the adapter to the 5’ of the template DNA. It is highly advantageous to reduce the ligation of the 3’ end of the adapter to the 5' of the template DNA since this can result in circular ligation products which are not compatible with subsequent steps leading to NGS library generation.
  • the use of a dephosphorylation step is in contrast to current NGS PCR amplification approaches which incorporate phosphorylation of template DNA. Instead, the present invention preferably makes use of dephosphorylated template DNA which results in aRNA production.
  • the present invention preferably makes use of dephosphorylated target DNA which results in improved aRNA production.
  • the method comprises a multiplex method.
  • a multiplex method may utilise sample bar codes allowing for the multiplexing of tens to thousands of samples following the ligation of the adapter oligonucleotide and the template DNA.
  • the method may further comprises a restriction enzyme digestion of the template DNA prior to the ligation of step ii).
  • restriction enzymes are suitable to perform the restriction enzyme digestion which are known in the art.
  • the restriction enzyme may produce“blunt ends” or“sticky ends”.
  • Restriction enzymes suitable to produce blunt ends may be selected from MLyl, BsuRI, Haelll, EcoRV, Smal.
  • Restriction enzymes suitable to produce sticky ends may be selected from NIAIII, Hsp92ll, Hinl ll, Mspl. Wherein sticky ends are produced by the restriction enzymes the target DNA may then undergo end repair and/or addition of an A tail.
  • a restriction enzyme which produces blunt ends may be used, more preferably MLyl or BsuRI may be used.
  • Step v) of the method of the present invention comprises transcribing the template DNA by introducing an RNA polymerase to the adapter oligonucleotide-template DNA molecule, to generate an aRNA product.
  • an RNA polymerase As described above, there are many RNA polymerases which could be utilised in the method of the present invention, such as T3 RNA polymerase, SP6 RNA polymerase and T7 RNA polymerase. However, in a preferred embodiment, the RNA polymerase may comprise a T7 RNA polymerase.
  • RNA polymerase By introducing an RNA polymerase to the adapter oligonucleotide-template DNA molecule to transcribe the template DNA, linear amplification of each original DNA strand is achieved. Unlike PCR approaches, which require an initially potentially error prone polymerisation step prior to amplification, around 1000 aRNA copies are made directly from each input strand, providing a means to reduce sequencing errors and increase specificity and sensitivity when detecting rare mutations in DNA mixtures.
  • the method further comprises the step of:
  • the method may further comprise the steps of: vi) converting the aRNA product to cDNA; and
  • the method of the present invention may be a method for amplifying template DNA.
  • the present invention therefore also provides a method for amplifying template DNA, the method comprising: i) providing template DNA and an adapter oligonucleotide comprising an RNA polymerase promoter sequence, wherein the adapter oligonucleotide is configured so that a 5’ end of the adapter oligonucleotide can be ligated to a 3’ end of the template DNA;
  • a secondary oligonucleotide comprising an RNA polymerase promoter sequence, wherein the sequence of the secondary oligonucleotide is complementary to the sequence of the adapter oligonucleotide;
  • cDNA refers to complementary DNA and is used to refer to DNA synthesised from single-stranded RNA, for example in a reaction catalysed by the enzyme reverse transcriptase.
  • Step vi) may first comprise ligating a priming site to the aRNA followed by reverse transcription using the priming site and a reverse transcriptase.
  • step vi) may comprise utilising reverse transcriptase and random oligonucleotide primers or utilising reverse transcriptase and target specific oligonucleotide primers.
  • any suitable reverse transcriptase enzyme could be utilised, for example the bacterial ret reverse transcriptase, the retroviral reverse transcriptases including RSV reverse transcriptase from Rous sarcoma virus, HIV-1 reverse transcriptase from human immunodeficiency virus type 1 , MMLV reverse transcriptase from Moloney murine leukemia virus and AMV reverse transcriptase from avian myeloblastosis virus.
  • the reverse transcriptase comprises of an optimised commercial cloned reverse transcriptase such as the mutant MMLV Superscript IV.
  • the method to convert the aRNA to cDNA may be performed in a number of ways. Conversion of aRNA to cDNA may be performed via ligation of a priming site to the aRNA. The ligation may be followed by reverse transcription. Conversion of aRNA to cDNA may be performed via single stranded ligation of a priming site to the aRNA. The single stranded ligation may ligate a unique NGS primer to the target aRNA. Conversion of aRNA to cDNA may be performed via splinted double stranded ligation to aRNA. The splinted double stranded ligation may ligate a unique NGS primer to the target aRNA. Conversion of aRNA to cDNA may be performed using random NGS primer. Conversion of aRNA to cDNA may be performed using target specific NGS primer.
  • Step vii) may comprise amplification in a next generation sequencing (NGS) protocol.
  • NGS next generation sequencing
  • the present invention as defined above is compatible with all of the current NGS platforms including systems which rely on clonal amplification of adapter-ligated libraries such as lllumina and Ion Torrent as well as single molecule sequencing platforms such as Pacific Bioscience and Oxford Nanopore.
  • Both optics-based sequencers which use visible light, collecting photons from arrays to form a visual image of bases incorporated during polymerization (e.g. lllumina) and semiconductor sequencing which rely on the detection of hydrogen ions released during the polymerisation of DNA (e.g. Ion Torrent) rely on the inclusion of specific adapter sequences which can be readily incorporated into the method of the present invention.
  • single molecule sequencing platforms such as Pacific Bioscience and Oxford Nanopore rely on addition of sequences that facilitate sequencing and these can also be readily incorporated into the method of the present invention.
  • the method can be used with both capture based NGS, where specific sequences are enriched following generation of a genome wide library, and direct PCR amplicon NGS.
  • the method of the present invention includes providing an adapter oligonucleotide comprising an RNA polymerase promoter sequence, wherein the adapter oligonucleotide is configured so that a 5’ end of the adapter oligonucleotide can be ligated to a 3’ end of the template DNA.
  • the 5’ end of the adapter oligonucleotide is ligated to the 3’ end of the template DNA to produce an adapter oligonucleotide-template DNA molecule and a secondary oligonucleotide is then annealed to the adapter oligonucleotide-template DNA molecule.
  • the adapter oligonucleotide comprises complementary regions which are capable of annealing to one another to form a secondary structure, preferably a circular or hairpin loop structure.
  • a secondary structure preferably a circular or hairpin loop structure.
  • the skilled person will appreciate how such an adapter could be designed.
  • the use of a circular or hairpin loop structure simplifies the method of the present invention in that by using an adapter with such a structure, it is not necessary to determine the optimum molar balance which becomes necessary when multi molecule adapters are utilised.
  • step iv) of the method may further comprise the step of denaturing the adapter oligonucleotide prior to annealing the secondary oligonucleotide to the adapter oligonucleotide.
  • the adapter oligonucleotide may comprise a number of additional sequence features which may be useful in further analysing the target DNA which has been amplified, such as molecular bar code, sample bar codes, a NGS sequence and/or a T tail.
  • the adapter oligonucleotide may further comprise a single region which is complementary, or partially complementary, to the stick ends produced by the restriction enzyme digestion.
  • the adapter oligonucleotide may further comprise a molecular barcode sequence.
  • a molecular bar code sequence may comprise, for example, a sequence of random bases, for example, 5, 6, 7, 8, 9 or 10 bases.
  • the molecular barcode sequence may include 6-8 bases. The molecular barcode provides a unique bar code for each adapter oligonucleotide that in turn will be incorporated into each adapter oligonucleotide-template DNA molecule.
  • RNA polymerisation of each adapter oligonucleotide-template DNA molecule will produce aRNA molecules each harbouring the same molecular bar code sequence as well as the same input DNA sequence enabling bioinformatics identification of all aRNA molecules originating from each single initial ligation event.
  • a consensus sequence can be built up that avoids identifying errors present in single aRNA molecules which may arise due to polymerase error. This will therefore increase the specificity and sensitivity which is particularly important for detecting rare mutations in DNA mixtures.
  • molecular barcodes along with a linear amplification step the error obtained using NGS is expected to be reduced by one to three orders of magnitude.
  • the adapter oligonucleotide may further comprise a sample barcode.
  • a sample barcode may comprise, for example, a sequence of random bases, for example, 5, 6, 7, 8, 9 or 10 bases.
  • each adapter may comprise a unique sample bar code which is ligated to a sample from a particular source. Following ligation of the adapter oligonucleotide and the template DNA, samples could be combined for subsequent steps, thereby improving efficiency. Post-sequencing information, for example, could be identified for each sample bioinformatically, through identifying the sample barcode.
  • the adapter oligonucleotide may further comprise a NGS sequence.
  • the adapter oligonucleotide may further comprise a T-tail.
  • the template DNA may comprise an A-tail, encouraging the adapter oligonucleotide and template DNA to anneal (due to the corresponding A- and T-tails) such that they are in proximity for subsequent ligation.
  • the template DNA is double-stranded.
  • the adapter oligonucleotide may only anneal to a single strand of said double-stranded DNA.
  • the adapter oligonucleotide may further comprise an overhang of oligonucleotides. This may be given the designation NNNNNNNN.
  • the overhang may be positioned after one of the complementary regions which are capable of annealing to one another.
  • the overhang may comprise a random sequence of oligonucleotides, or the sequence may be selected to be complementary to the target DNA.
  • the overhang may be 5’ or 3’.
  • the adapter oligonucleotide forms a circular or hairpin secondary structure and comprises an overhang.
  • the oligonucleotide overhang may comprise about 6 to about 12 oligonucleotides, preferably about 8 to about 10 oligonucleotides, more preferably about 8 oligonucleotides.
  • This format of the adapter oligonucleotide may be particular suitable for use in single strand ligation.
  • the template DNA may be derived from a biological sample.
  • the biological sample may comprise blood.
  • the template DNA may comprise cfDNA, gDNA and/or single cell DNA.
  • suitable commercial kits are available for obtaining template DNA, including cfDNA, gDNA or single cell DNA.
  • suitable kits include for example QIAamp DNA Blood Mini Kit (Qiagen), QIAamp circulating nucleic acid kit (Qiagen) and MagMAX Cell-free DNA Isolation Kit (Thermo Fisher Scientific).
  • the template DNA is double-stranded DNA or single-stranded DNA.
  • Ligation to single-stranded DNA can incorporate improved ligation conditions including use of optimised ligase (Gansauge and Meyer, 2013) or primer design incorporating ligation guides or splinter oligonucleotides (Gansauge et al, 2017; Kwok et al, 2013).
  • an adapter oligonucleotide for producing an aRNA product from a DNA template, the adapter oligonucleotide comprising an RNA polymerase promoter sequence, wherein the adapter oligonucleotide is configured so that a 5’ end of the adapter oligonucleotide can be ligated to a 3’ end of a template DNA.
  • an adapter oligonucleotide for amplifying a DNA template, the adapter oligonucleotide comprising an RNA polymerase promoter sequence, wherein the adapter oligonucleotide is configured so that a 5’ end of the adapter oligonucleotide can be ligated to a 3’ end of a template DNA.
  • the adapter of the present invention may be for use in the method of the present invention, as defined above.
  • the RNA polymerase promoter sequence may comprise a T7 RNA polymerase promoter sequence, and such a sequence may comprise SEQ ID N0.1 or a fragment or variant thereof.
  • the RNA polymerase promoter sequence may consist of SEQ ID N0.1.
  • the adapter may comprise complementary regions which are capable of annealing to one another to form circular or hairpin loop structure.
  • the adapter may comprise a number of additional sequence features which may be useful in further analysing the target DNA which has been amplified, such as molecular bar code, sample bar codes, a NGS sequence and/or a T tail.
  • the adapter may be in a buffered (or other) solution so as to enable storage and transportation of the adapter.
  • a buffered (or other) solution so as to enable storage and transportation of the adapter.
  • the skilled person will appreciate suitable solutions in which the adapter may be present.
  • the adapter may be in vial or other vessel suitable for storage, transportation and mixing with other reagents if desired.
  • the adapter is for use as a research reagent/tool.
  • kits for producing an aRNA product from a DNA template comprising:
  • an adapter oligonucleotide comprising an RNA polymerase promoter sequence, wherein the adapter oligonucleotide is configured so that a 5’ end of the adapter oligonucleotide can be ligated to a 3’ end of a template DNA;
  • a secondary oligonucleotide comprising an RNA polymerase promoter sequence, wherein the sequence of the secondary oligonucleotide is complementary to the sequence of the adapter oligonucleotide.
  • kit for amplifying a DNA template comprising:
  • an adapter oligonucleotide comprising an RNA polymerase promoter sequence, wherein the adapter oligonucleotide is configured so that a 5’ end of the adapter oligonucleotide can be ligated to a 3’ end of a template DNA; and ii) a secondary oligonucleotide comprising a RNA polymerase promoter sequence, wherein the sequence of the secondary oligonucleotide is complementary to the sequence of the adapter oligonucleotide.
  • the RNA polymerase promoter sequence in the adapter oligonucleotide comprises a T7 RNA polymerase promoter sequence with a sequence comprising SEQ ID N0.1 or a fragment or variant thereof.
  • the RNA polymerase promoter sequence in the adapter oligonucleotide consists of SEQ ID N0.1.
  • the RNA polymerase promoter sequence in the secondary oligonucleotide comprises a T7 RNA polymerase promoter sequence with a sequence comprising SEQ ID NO.2 or a fragment or variant thereof.
  • the RNA polymerase promoter sequence in the adapter oligonucleotide consists of SEQ ID NO.2.
  • the adapter oligonucleotide of the kit may comprise complementary regions which are capable of annealing to one another to form circular or hairpin loop structure as described above in relation to the method of the present invention.
  • the adapter oligonucleotide of the kit may comprise a number of additional sequence features which may be useful in further analysing the target DNA which has been amplified, such as molecular bar code, sample bar codes, a NGS sequence and/or a T tail as described above in relation to the method of the present invention.
  • the adapter oligonucleotide and/or secondary oligonucleotide of the kit may be in a buffered solution.
  • the skilled person will appreciate suitable solutions in which the adapter may be present.
  • a method for detecting a genetic variant or genetic signature in template DNA comprising amplifying template DNA in the method as herein above described and analysing the amplified cDNA for the genetic variation or signature.
  • the method of the present invention can be used to identify a wide range of genetic signatures including the presence of specific point mutations, mutational signature and structural alterations including copy number gains and losses.
  • the information generated can be used to identify mutations linked to specific therapy, monitor disease, predict clinical outcomes and response to therapy. It is also envisaged that the invention will be particularly suited to identifying differences in DNA methylation that are related to the detection of tumour DNA in patient blood samples. Differences in DNA methylation may comprise differences in total methylation or DNA methylation patterns or signatures (such as described in Shen, S.Y., et. ai, (2016) and Shina. A. A. I. et. ai, (2018)).
  • the method of the present invention can be used to identify the presence, variations, or signatures, of ctDNA in the blood of cancer patients (or patients who are at a high risk of developing cancer or having a reoccurrence of cancer) and can also be used to identify the presence, variation or signature of circulating tumour cells (CTCs).
  • the method can be used to detect sequence variants that are known to be specifically associated with various types of tumours or harbour copy number patterns linked to the presence of tumours and their progression.
  • a method of preparing template DNA for amplification comprising dephosphorylating the template DNA.
  • dephosphorylating the template DNA is achieved using a phosphatase enzyme.
  • the method preferably removes the 5’ phosphate of the template DNA.
  • the phosphatase enzyme may comprise one or more selected from the following: Shrimp Alkaline Phosphatase; Antarctic Phosphatase; Calf Intestinal Alkaline Phosphatase; and/or or FastAP Thermosensitive Alkaline Phosphatase.
  • the phosphatase enzyme comprises a heat labile phosphatase such as FastAP Thermosensitive Alkaline Phosphatase.
  • a phosphatase enzyme for dephosphorylating template DNA prior to amplification or modification prior to amplification.
  • the use will preferably be to remove the 5’ phosphate of the template DNA.
  • the phosphatase enzyme may comprise one or more selected from the following: Shrimp Alkaline Phosphatase; Antarctic Phosphatase; Calf Intestinal Alkaline Phosphatase; and/or or FastAP Thermosensitive Alkaline Phosphatase.
  • the phosphatase comprises a heat labile phosphatase such as FastAP Thermosensitive Alkaline Phosphatase.
  • a method for amplifying template DNA comprising: i) providing template DNA and an adapter oligonucleotide, wherein the adapter oligonucleotide is configured so that a 5’ end of the adapter oligonucleotide can be ligated to a 3’ end of the template DNA;
  • the method may further comprise transcribing the template DNA by providing a RNA polymerase promoter sequence in the adapter oligonucleotide sequence and introducing an RNA polymerase to the adapter oligonucleotide-template DNA molecule under conditions so to generate an aRNA product, and to converting the aRNA product to cDNA and subsequently amplifying the cDNA.
  • dephosphorylating the template aRNA or DNA is achieved using a phosphatase enzyme.
  • the method preferably removes the 5’ phosphate of the template aRNA or DNA.
  • the phosphatase enzyme may comprise one or more selected from the following: Shrimp Alkaline Phosphatase; Antarctic Phosphatase; Calf Intestinal Alkaline Phosphatase; and/or or FastAP Thermosensitive Alkaline Phosphatase.
  • the phosphatase enzyme comprises a heat labile phosphatase such as FastAP Thermosensitive Alkaline Phosphatase.
  • Figure 1 is a schematic diagram for amplifying cfDNA as described in Example 1 ;
  • Figure 2 is a schematic diagram for amplifying genomic and circulating cell-free DNA as described in Example 2 (where steps 4-6 are carried out as depicted in Figure 1);
  • Figure 3 is a schematic diagram for amplifying genomic and circulating cell-free DNA as described in Example 3, where steps 4-6 are carried out as depicted in Figure 1 ;
  • Figure 4 shows the result of the cfDNA ligation kit selection experiments as described in Example 4 for a dsDNA cfDNA T7 library preparation comparing NEB quick, NEB ultra and KAPA ligases, where Figure 3A shows a photograph of a electrophoretic gel and Figure 4B is a graph;
  • Figure 5 shows the result of the cfDNA ligation optimisation experiments as described in Example 5, where the experiments were conducted between 4 - 16°C and where Figure 5A shows a photograph of a electrophoretic gel and Figure 5B is a graph;
  • Figure 6 shows the result of the cfDNA ligation optimisation experiments as described in Example 6, where the experiments were conducted between 12 - 20°C and where Figure 6A shows a photograph of a electrophoretic gel and Figure 6B is a graph;
  • Figure 7 shows the result of the cfDNA ligation optimisation experiments as described in Example 7, where Figure 7A shows a photograph of a electrophoretic gel illustrating the results of the KAPA ligase T7 library preparation and Figure 7B is a graph;
  • Figure 8 shows the result of NGS analysis of HNV and cancer patient cfDNA as described in Example 8, where Figure 8A shows a photograph of a electrophoretic gel and Figure 8B shows plots of genome wide copy number changes;
  • Figure 9 shows that the present DNA amplification method is compatible with DNA methylation analysis.
  • Figure 9A shows a photograph of an electrophoretic gel showing index PCR products from the present method, lanes 2 and 3 show input (no enrichment control) pool 1 and 2 (In1 and In2) and lanes 3 and 4 show methylation-enriched pool 1 and 2 (Me1 and Me2).
  • Figure 9B shows the results of sample bar code demultiplexing;
  • Figure 10 shows that the present DNA amplification method is compatible with DNA methylation analysis and delivers improved results compared to a non-UMI-based NGS library preparation method.
  • Figure 10A shows Methylation Enrichment QC which demonstrates greater enrichment using the present method (T7 pool and individual) compared to non-UMI based method (current method).
  • Figure 10B shows Methylation background QC and demonstrates a reduction in background noise with the present method (T7 pool and individual) compared to non-UMI based method (current method). More than double the number of reads in the non-UMI based method (current method) do not contain a CpG.
  • Figure 10C shows PCA plot separates tumour and normal samples.
  • Figure 10D shows HOXA9 tumour vs. normal samples and demonstrates that the present method can identify tumour-specific methylation;
  • Figure 11 shows comparative data of the present DNA amplification method (T7) vs a commercial amplification method using UMI NGS adapters (IDT).
  • Figure 11A shows analysis of the methylation enrichment, the present method shows improvement in enrichment over the commercial method.
  • Figure 11 B shows analysis of the methylation background, the present method shows a significant reduction in the amount of background compared to the commercial method.
  • Figure 11C shows that the present method has enhanced sensitivity over the commercial method;
  • Figure 12 shows amplification performed on DNA digested using three restriction enzymes, MLyl, BsuRI and NIAIII. Both MLyl and BsuRI produce blunt ends and NIAIII produces a 3’ overhang.
  • Figure 12A shows the restriction sites recognised by MLyl, BsuRI and NIAIII and a photograph of an electrophoretic gel of the DNA post digestion with each of the restriction enzymes individually.
  • Figure 12B shows a photograph of an electrophoretic gel of the digested DNA after performing amplification using the present method.
  • Figure 12C shows a schematic of the method for amplifying genomic and single cell gDNA comprising the steps of: Step 1 : prepare gDNA by utilising a MLyl or BsuRI restriction enzyme digest, resulting in blunt end DNA fragments, which are then A-tailed. Step 2: anneal the oligonucleotide so as to form a circular adapter. Step 3: ligates the circular adapter to target DNA;
  • Figure 13 shows the results of the in-silico folding of the adapter using Quickfold software. This demonstrates the complementary regions of the adapter are capable of self-annealing and forming circular secondary structure;
  • Figure 14 demonstrates the suitability of pooling multiple samples after ligation. Pooled NGS library samples were sequenced and then demultiplexed;
  • Figure 15 shows conversion of aRNA to cDNA.
  • Figure 15A demonstrates the conversion of aRNA to cDNA using the ligation approach or a random primer approach.
  • Figure 15B shows optimisation of the conversion of aRNA to cDNA using the ligation approach or a random primer approach.
  • For the ligation approach one sample has been treated with phosphatase and one sample has not been treated with phosphatase prior to the ligation step.
  • For the random primer approach one sample has used 100 mM primer and one sample has used 50 pM primer.
  • Figure 15 C and D show schematics of the conversion of aRNA to cDNA using the ligation approach (15C) or a random primer approach (15D); and
  • Figure 16 shows amplification of cfDNA.
  • Figure 16A shows amplification of cfDNA using the present method with different input amounts of cfDNA.
  • Figures 16B and C show RNaseP qPCR quantification and RNaseP qPCR fold increase in aRNA relative to input DNA respectively.
  • Step 1 prepare target cfDNA to be A-tailed cfDNA and dephosphorylated.
  • Step 2 utilise a unique oligonucleotide sequence to form self-annealing circular adapter.
  • Step 3 ligates the circular adapter to target DNA. Ligation only occurs to one strand so as to enable separate independent sequencing of both upper and lower strands of the DNA.
  • Step 4 denatures and anneals a T7 RNA polymerase complementary oligonucleotide. This step does not require a 2nd polymerisation step to produce dsDNA and is therefore simpler and more efficient than prior art DNA polymerase amplification.
  • Step 5 employs a T7 RNA polymerase reaction on the complementary oligonucleotide and amplify the strand.
  • all amplified aRNA products are generated by producing up to 1 ,000 aRNA copies of each original unaltered strand.
  • Step 6 converts the aRNA into cDNA in preparation for sequencing. It will be apparent to the skilled person that this step will be dependent upon which sequencing platform will be utilised and would typically be selected from one of the following outlined below:
  • Step 6a converts aRNA to cDNA via ligation to aRNA. This has the benefit of preserving the original fragment length, but does involve an extra possible inefficient step in the process.
  • Step 6b converts aRNA to cDNA using random NGS primer. This has the benefit of being simple and efficient, but the original fragment length is lost.
  • Step 6c converts aRNA to cDNA using target specific NGS primer. This has the benefit of being highly specific, but is limited to the target.
  • Example 2 Method for amplifying genomic and circulating cell-free DNA
  • a method for amplifying genomic and circulating cell-free DNA is schematically shown in Figure 2 and has the following steps 1 - 3:
  • Step 1 prepare gDNA by utilising a Hsp92ll restriction enzyme digest.
  • Step 2 anneal the oligonucleotide so as to form a circular adapter.
  • Step 3 ligates the circular adapter to target DNA. Since the adapter T7_U1_ Hsp92ll3c3 contains only 3 of the 4 bases of the overhang, this reduces primer dimer. Furthermore, adapter T7_U1_ Hsp92ll2c3 contains only 2 of the 4 bases of the overhang - further reducing primer dimer.
  • the method may additionally comprise steps 4 - 6 (not shown):
  • Step 4 denature and anneal the T7 RNA polymerase complementary oligonucleotide.
  • Step 5 employs a T7 RNA polymerase reaction on the complementary oligonucleotide and amplify the strand.
  • Step 6 converts the aRNA into cDNA in preparation for sequencing. It will be apparent to the skilled person that this step will be dependent upon which sequencing platform will be utilised and would typically be selected from one of the following outlined below:
  • Step 6a converts aRNA to cDNA via ligation to aRNA. This has the benefit of preserving the original fragment length, but does involve an extra possible inefficient step in the process.
  • Step 6b converts aRNA to cDNA using random NGS primer. This has the benefit of being simple and efficient, but the original fragment length is lost.
  • Step 6c converts aRNA to cDNA using target specific NGS primer. This has the benefit of being highly specific, but is limited to the target.
  • FIG. 15C shows step 6a(i) involves adding a priming site to aRNA via ligation to U2 oligonucleotide to ligate U2 ligation adapter to aRNA.
  • the next steps would be the conversion of aRNA to full length cDNA using a primer complementary to U2 oligo and the formation of a NGS library with full length cDNA.
  • FIG. 15C shows step 6a(ii) involves converting aRNA to cDNA via improved ligation to aRNA.
  • a U2 ligation adapter is ligated to aRNA.
  • the rationale is that the NN adapter promotes annealing to aRNA thereby improving efficiency of ligation compared to U2 oligonucleotide alone (as set out in step 6a above).
  • the next steps would be the conversion of aRNA to full length cDNA using primer complementary to U2 oligonucleotide and the formation of a NGS Library with full length cDNA.
  • FIG. 15D shows step 6b(i) involves preparing cDNA from aRNA using a U2 random primer. aRNA is converted to cDNA in a reverse transcriptase reaction. The next steps would be the formation of a NGS Library direct with cDNA from the reverse transcriptase reaction.
  • FIG. 15D shows step 6b(ii) involves promoting full length reverse transcriptase products using a random primer adapter.
  • aRNA is converted to cDNA in a reverse transcriptase reaction.
  • the rationale is that the U2 Random Primer adapter promotes priming from the end of each aRNA by sterically blocking/reducing internal priming and this will provide more full length cDNA than U2 Random Primer alone (as set out in step 6c above).
  • the next steps would be the formation of a NGS library direct with cDNA from the reverse transcriptase reaction.
  • Example 3 Method for amplifying single stranded genomic and circulating cell-free DNA
  • a method for amplifying single stranded genomic and circulating cell-free DNA, according to the present invention, is schematically shown in Figure 3 and has the following steps:
  • Step 1 prepare single stranded DNA by denaturation (heat or chemical).
  • Step 2 anneal the oligonucleotide so as to form a circular adapter.
  • Step 3 anneal and ligate the circular adapter to target DNA.
  • Step 4 denature and anneal a T7 RNA polymerase complementary oligonucleotide. This step does not require a 2nd polymerisation step to produce dsDNA and is therefore simpler and more efficient than prior art DNA polymerase amplification.
  • Step 5 employs a T7 RNA polymerase reaction on the complementary oligonucleotide and amplify the strand. In this step, all amplified aRNA products are generated by producing up to 1 ,000 aRNA copies of each original unaltered strand.
  • Step 6 converts the aRNA into cDNA in preparation for sequencing. It will be apparent to the skilled person that this step will be dependent upon which sequencing platform will be utilised and would typically be selected from one of the following outlined below:
  • Step 6a converts aRNA to cDNA via ligation to aRNA. This has the benefit of preserving the original fragment length, but does involve an extra possible inefficient step in the process.
  • Step 6b converts aRNA to cDNA using random NGS primer. This has the benefit of being simple and efficient, but the original fragment length is lost.
  • Step 6c converts aRNA to cDNA using target specific NGS primer. This has the benefit of being highly specific, but is limited to the target.
  • the experiments also showed clear aRNA products from cfDNA with qPCR indicating 25-80x amplification (not shown) and NTC shows primer aRNA products overlapping in size with cfDNA aRNA products.
  • Step 1 was to polish and add a dA tail to the DNA.
  • PCR tubes all samples were adjusted to 35 mI with nuclease-free water. To each 35 mI of sample the following products were added (NOTE: defrost and place the reagents on ice, then prepare the tailing reaction on the bench at room temp):
  • Step 2 was to ligate the adapter to tailed DNA under the following conditions (NOTE: When Step 1 is running prepare T7dT_U1_Lcf1 oligo):
  • a ligation master mix was then prepared on ice as followed:
  • Step 3 was to clean-up post ligation:
  • T7_prim1 oligo was prepared as follows:
  • Step 5 was the T7 aRNA production.
  • a T7 master mix was prepared as follows:
  • T7 master mix 12.5 mI of the T7 master mix was then dispensed into six PCR tubes and the tubes were placed into the thermocycler on hold at 37°C. The master mix was left in the block for 1 min to reach 37°C. Then 12.1 mI of the pre-warmed T7 master mix was transferred to each tube containing the annealed T7_Prim1/ligation product, which is already on hold at 37°C. 2.4 mI of T7 polymerase was then added to each tube and mixed by pipetting 10 times.
  • a T7 run off positive control reaction was prepared as follows:
  • the positive control tubes were then placed on the block on hold at 37°C and left to run overnight with the other reactions.
  • Step 6 involved the clean-up post aRNA production.
  • An Ampure bead clean was performed as follows (exclude the positive control):
  • Table 10 10 pi of eluate was transferred to a fresh PCR tube and placed on ice. In a QC step, 1 pi of purified aRNA was run on a gel to assess output (along with 5 mI of 1 :100 diluted positive control reaction).
  • Step 7 was to convert aRNA to cDNA.
  • the following oligonucleotide was prepared:
  • the oligonucleotide was then mixed with the aRNA on ice at the following concentrations/volumes:
  • the denaturing and annealing were performed on a thermocycler using the following conditions:
  • thermocycler The mixture was then placed on a thermocycler and run using the following programme:
  • Step 8 involved the clean-up post aRNA RT.
  • An Ampure bead clean-up was performed as follows:
  • Step 9 involves the NGS PCR.
  • a NGS PCR master mix was prepared as follows:
  • Step 10 involved the clean-up post NGS PCR. Ampure bead clean was performed as follows:
  • Step 11 involved NGS. Libraries were quantified using Kapa qPCR according to FRM-94. Libraries were pooled in equimolar or amounts to generate a composite library sample. Composite library was quantified using KAPA qPCR according to FRM-94. The composite library was denatured and diluted to 12.5 pM and supplemented with 10% PhiX (2 pM final cone.). The NGS run was performed on the Miseq using Miseq Kit (300 cycles) and paired end sequencing.
  • Table 19 below and Figure 6 shows the results of a T7 cfDNA Library which was run on an electrophoretic gel and shows very high banding with a library prep loading of 25 ng, but good banding too at 5.845 ng.
  • Step 1 end repair and dA-tailing
  • sample set is a dilution series of sheared gDNA from SCLC cell line H446 diluted in sheared gDNA from HNV PBMCs as shown below in Table 20.
  • Step 2 Dephosphorylation of the DNA samples was performed using Therma AP fast phosphatase. To each sample 5mI_ of AP fast phosphatase (1 U/pL) was added. Following mixing the samples were placed into PCR block and the following conditions applied (as detailed below in Table 23):
  • Step 3 preparation of the T7dT_U1_LcfX oligo hairpin for ligation
  • the annealed T7 oligo and subsequently the master mix were held on ice until the start of ligation.
  • the ligation master mix was prepared as follows (as detailed in Table 26):
  • T7dT_U1_Lcf oligo 2.2 mM was added to the dephosphorylated DNA and mixed thoroughly. Then 31.5 mI_ of master mix was added to dephosphorylated DNA, and mixed. Ligations were placed into the PCR machine running at 20 °C overnight, with heated lid off.
  • Step 5 preparation of the sample pool is shown below in Table 27.
  • Table 28 45mI_ of eluate was used for methylation pull down and 5 mI_ of the eluate was used as a no- methylation enrichment control.
  • Methylated DNA enrichment was performed as per the manufacturer’s protocol.
  • MBD2-Fc was prebound to Protein A magnetic beads.
  • a mix of protein and beads was prepared containing the following ingredients (as detailed in Table 29 below):
  • Capture of the methylated CpG DNA was performed using the components as detailed in Table 30.
  • Step 7 T7 amplification of methylation-enriched tumour DNA to aRNA was performed according to Table 31 below.
  • Table 33 For T7_prim1 dilution to 0.8 mM from 100pM stock. Dilute 1 :10 in TE to make 10 pM dilution, then 2pl of the 1 :10 dilution (10 pM) and 23pL H20.
  • Step 12 ssLigation
  • RT oligo was prepared as follows. RT_U2P_primer (100 mM) (GACGTGTGCTCTTCCGATCT) was used.
  • RT_U2P_primer 100 mM was added to each adapter-ligated aRNA sample. The samples were then denatured and annealed using the following conditions (as detailed in Table 44).
  • Table 44 The RT master mix was prepared as follows (as detailed in Table 45):
  • Step 15 indexing PCR
  • the master mix for the indexing PCR was prepared as follows (as detailed in Table 48 below).
  • Table 48 17.5mI_ of the PCR master mix was added to 30mI_ of purified RT reactions. 2.5mI_ of the indexing oligo was added. Then the following PCR conditions were applied (as detailed in Table 49):
  • Example 10 Comparison of methylation enriched libraries prepared by the present method and a commercially available method
  • the adapter ligated DNA was enriched for methylated DNA using the EpiMark Methylated DNA Enrichment Kit (New England BioLabs Inc.). Methylation enriched DNA was amplified in an NGS PCR, which added NGS adapters and a sample index. Samples were pooled and quantified by KAPA qPCR, and then run on an lllumina NextSeq instrument. Individual methylation enriched NGS libraries were prepared for each sample using the T7 method described in the present invention. In addition, samples were also pooled after the first ligation of the T7 circular adapter and this pool of samples was enriched for methylated DNA and NGS libraries generated using the T7 method described in the present invention.
  • Figure 10 A and B show that the T7 method results in a much higher enrichment of methylated DNA as well as reducing the background of non-methylated DNA (reads that do not contain a CpG). Comparison of the T7 method run on a pool of samples compared to individual samples shows that pooling the samples results in more uniform data.
  • Figure 10C shows that tumour and matched normal samples can be separated based on the methylation patterns determined by the T7 method.
  • Figure 10D shows that the method is picking out known tumour-specific methylation, that is not detected in the matched normal tissue sample.
  • Example 11 Further comparison of methylation enriched libraries prepared by the present method and a commercially available method
  • the dilution series was 100% cell line, 5% cell line, 2.5% cell line, 1.0% cell line, 0.1 % cell line, 0.01 % cell line, 0.001% cell line and 0% cell line (100% HNV PBMC)
  • the commercial UMI based NGS library preparation method started with end repair and A tailing of the DNA, followed by ligation of the xGen Dual Index UMI Adapters from IDT (Integrated DNA Technologies) using KAPA-Hyper DNA ligase and KAPA-Hyper ligation buffer.
  • the adapter ligated DNA was pooled and enriched for methylated DNA using the EpiMark Methylated DNA Enrichment Kit (New England BioLabs Inc.). Methylation enriched DNA was amplified in an NGS PCR.
  • FIG. 11 A and B show that the T7 method results in a slightly higher enrichment of methylated DNA, but greatly reduces the background of non-methylated DNA (reads that do not contain a CpG).
  • Figure 11C shows that in windows (of 200bp) that are methylated in the cell line but not in the HNV PBMCs, there are more reads in the T7 method at lower dilutions of the cell line, indicating that the T7 method is more sensitive than the commercial UMI adapters.
  • Example 14 Formation of circular adapter using various buffers.
  • TE buffer Invitrogen, Fisher Scientific
  • TNE buffer Tris 100mM, NaCI 500mM and EDTA 10mM
  • JENA buffer 5 Jena Bioscience
  • the TNE buffer and JENA buffer 5 were also tested with increasing amounts of PEG present in the reaction.
  • aRNA products formed following T7 RNA transcription were analysed by agarose gel and showed that the hairpin formation works in all buffer conditions (data not shown).
  • Table 52 below shows the number of copies of aRNA produced under each condition as quantified by RNase P qPCR.
  • aRNA was generated as described in steps 1-6 of Example 8.
  • the following ligation master mix was prepared according to the conditions outlined in Table 55 below;
  • Table 57 The components outlined in Table 57 were denatured and annealed using the conditions outlined in Table 58 below.
  • the RT reaction master mix was prepared as outlined in Table 59.
  • Table 59 8mI_ of RT reaction master mix was added to the aRNA/U2P_primer mix, followed by 1 mI_ of Superscript IV. Samples were placed in the PCR block and the conditions outlined in Table 60 were applied
  • the RT reaction products were cleaned up using Ampure bead clean up using the conditions outlined in Table 61. Products were eluted in low EDTA TE buffer.
  • Step 10 Indexing PCR
  • the indexing PCR master mix was prepared using the conditions outlined in Table 62.
  • Samples were then clean-up using Ampure bead clean up and products eluted in low EDTA TE buffer. Samples were stored before quantification and NGS.
  • Example 17 Amplification performed with various amounts of cfDNA.
  • T7RNA_P T7 RNA Polymerase promoter sequence
  • AGATCGGAAGAGCACACGTC modified with a phosphate group at 5' and a C3 spacer (blocker) at the 3' U2P RT 1 (SEQ ID N0.6)

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Abstract

La présente invention concerne un procédé d'amplification d'ADN de matrice, le procédé comprenant : i) l'utilisation d'ADN de matrice et d'un oligonucléotide adaptateur comprenant une séquence de promoteur d'ARN polymérase, l'oligonucléotide adaptateur étant configuré de telle sorte qu'une extrémité 5' de l'oligonucléotide adaptateur peut être ligaturée à une extrémité 3' de L'ADN de matrice; ii) la ligature de l'extrémité 5' de l'oligonucléotide adaptateur à l'extrémité 3' de l'ADN de matrice pour produire une molécule d'oligonucléotide adaptateur-ADN de matrice; iii) fournir un oligonucléotide secondaire comprenant une séquence de promoteur d'ARN polymérase, la séquence de l'oligonucléotide secondaire étant complémentaire de la séquence de l'oligonucléotide adaptateur; iv) le recuit de l'oligonucléotide secondaire à la molécule d'oligonucléotide adaptateur-ADN de matrice; et V) la transcription de l'ADN de matrice par l'introduction d'une ARN polymérase à la molécule d'oligonucléotide adaptateur-ADN de matrice, pour produire un produit d'ARNi. Le procédé de la présente invention peut en outre comprendre vi) la conversion du produit ADNa en ADNc; et vii) l'amplification de l'ADNc. L'invention porte également sur des oligonucléotides adaptateurs une trousse et des procédés d'utilisation de telles thérapies.
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