WO2021224225A1 - Procédé - Google Patents

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Publication number
WO2021224225A1
WO2021224225A1 PCT/EP2021/061671 EP2021061671W WO2021224225A1 WO 2021224225 A1 WO2021224225 A1 WO 2021224225A1 EP 2021061671 W EP2021061671 W EP 2021061671W WO 2021224225 A1 WO2021224225 A1 WO 2021224225A1
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Prior art keywords
dna
sample
cross
dna fragments
hours
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PCT/EP2021/061671
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English (en)
Inventor
Joost F Swennenhuis
Erik C SPLINTER
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Cergentis B.V.
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Priority claimed from GBGB2006550.4A external-priority patent/GB202006550D0/en
Priority claimed from GBGB2010494.9A external-priority patent/GB202010494D0/en
Application filed by Cergentis B.V. filed Critical Cergentis B.V.
Publication of WO2021224225A1 publication Critical patent/WO2021224225A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor

Definitions

  • the present invention relates to the field of molecular biology.
  • the present invention relates to methods for recovering polynucleotides from preserved, cross-linked tissue samples - such as formalin-fixed, paraffin-embedded samples (FFPE).
  • FFPE formalin-fixed, paraffin-embedded samples
  • the invention relates to the sequencing of DNA, for example to strategies for determining a DNA sequence of a genomic region of interest.
  • the invention relates to the determination of the sequence of parts of a genome that are in a spatial configuration with each other.
  • tissue samples - such as formalin-fixed, paraffin-embedded samples (FFPE) - are a common sample type in both clinical and research settings.
  • FFPE paraffin-embedded samples
  • biopsies and surgical samples are prepared as FFPE tissue blocks. Accordingly, these samples provide a notable resource for determining genetic information; for example to identify the presence of mutations from e.g. a tumour sample.
  • fixation conditions applied during the production of such samples are often harsh and result in heavy cross-linking, including the generation of heavily cross-linked DNA.
  • Methods for reversing the cross-linking of DNA typically comprise incubation at an intermediate temperature, for example 65 °C, for a period of 4-12 hours.
  • reverse cross-linking may be performed by incubation at a higher temperature, for example 80 °C, for a short time period such as 60 minutes or for example 90 °C, for a short time period such as 30-60 minutes.
  • Efficient reversal of cross-linking is important for downstream molecular analysis. Incomplete reversal resulting in residual cross-links may inhibit downstream steps such as DNA yields following purification, reduce the integrity of the DNA recovered, and have major implications for the quality of downstream procedures.
  • the present invention is based on the inventors’ surprising determination that the use of particular conditions for the reversal of cross-linking in cross-linked samples, in particular heavily cross-linked samples such as FFPE tissue samples, provides an unexpected high quality of recovered DNA.
  • the conditions applied in the present methods unexpectedly result in both advantageous DNA yields and integrity, which are consider to result in higher quality data from downstream analysis.
  • the present invention provides a method for reversing the cross-linking of a sample comprising cross-linked polynucleotides, the method comprising incubating the sample at about 75 °C to about 87 °C for about 8 hours to about 24 hours.
  • the present invention relates to a method for extracting a polynucleotide from a sample comprising cross-linked polynucleotides; the method comprising incubating the sample at about 75 °C to about 87 °C for about 8 hours to about 24 hours.
  • the present methods may be applied in circumstances in which it is required to reverse the cross-linking of polynucleotides and/or extract cross-linked polynucleotides for downstream analysis such as PCR-based techniques.
  • the present invention provides a method for determining the presence or absence of a mutation in at least part of the sequence of a polynucleotide in a sample comprising cross-linked polynucleotides; the method comprising incubating the sample at about 75 °C to about 87 °C for about 8 hours to about 24 hours.
  • the polynucleotide may be DNA.
  • the present invention relates to a method for determining at least part of the sequences of DNA fragments from a sample of fragmented cross-linked DNA; which comprises the following steps: a) providing a sample of fragmented cross-linked DNA; b) optionally, further fragmenting the cross-linked DNA; c) optionally, repairing the ends of DNA fragments; d) ligating the cross-linked DNA fragments; e) reversing the cross-linking by incubating the sample at about 75 °C to about 87 °C for about 8 hours to about 24 hours; and f) determining at least part of the sequences of the DNA fragments.
  • the present invention relates to a method for determining at least part of the sequences of DNA fragments from a sample of fragmented cross-linked DNA; which comprises the following steps: a) providing a sample of fragmented cross-linked DNA; b) optionally, further fragmenting the crosslinked DNA; c) optionally, repairing the ends of DNA fragments to facilitate ligation; d) ligating the fragmented crosslinked DNA; e) reversing the crosslinking by incubating the sample at about 75 °C to about 87 °C for about 8 hours to about 24 hours; f) optionally fragmenting the DNA of step e), g) optionally, ligating the fragmented DNA of step e) or f) to at least one adaptor; h) optionally, (1) amplifying the ligated DNA fragments of step e) or f) comprising the target nucleotide sequence using at least one primer which hybridises to the target nucleotide sequence, or amplifying the
  • the present invention provides a method for determining the presence or absence of a mutation in a genomic region of interest comprising a target nucleotide sequence, comprising the steps of: a) providing a sample of fragmented cross-linked DNA; b) optionally, further fragmenting the crosslinked DNA; c) optionally, repairing the ends of DNA fragments to facilitate ligation d) ligating the fragmented crosslinked DNA; e) reversing the crosslinking by incubating the sample at about 75 °C to about 87 °C for about 8 hours to about 24 hours; f) optionally fragmenting the DNA of step e), g) optionally, ligating the fragmented DNA of step e) or f) to at least one adaptor; h) optionally, (1) amplifying the ligated DNA fragments of step e) or f) comprising the target nucleotide sequence using at least one primer which hybridises to the target nucleotide sequence, or amplifying the ligated
  • Figure 1 A) Yield of DNA isolations after overnight reverse crosslinking at temperatures ranging from 65 to 90°C using two FFPE samples: Sample 5694 and Sample 935. B) Agarose gel image of DNA isolated from FFPE tissues after overnight incubation at the indicated temperature (indicated at the top of the lanes).
  • Figure 2 A lOplex-multiplex end-point PCR on FFPE samples isolated after overnight reverse crosslinking at temperatures ranging from 65 to 90°C. Equal amounts of PCR products are run on a 2% agarose gel.
  • Figure 3 Relative yield of DNA isolations of three different FFPE tissues (5525, 769 and 961) treated according the indicated conditions. DNA yields were measured with Qubit and were normalized to the yield found at the 16 hour, 65°C condition.
  • Figure 4 Fold change in amplifiability relative to 16 h 65°C of 50 to 299 bp amplicons in DNA isolated from FFPE tissue 961 treated at different conditions.
  • Figure 5 Fold change in amplifiability relative to 16 h 65°C of 50 to 207 bp amplicons in DNA isolated from FFPE tissue treated at different reverse crosslinking conditions.
  • Figure 6 Fold change of amplifiability of three different FFPE tissues. A: 5525, B: 769 and C: 961. FFPE tissues were reverse crosslinked under different conditions indicated on the x-axes. Three different sized amplicons (81, 246 and 293bp) were tested for each sample.
  • Figure 7 DNA Yield multiplied by the fold change in amplifiability of the different reverse crosslinking conditions relative to 16h 65 °C for three different FFPE tissues and three different amplicon sizes.
  • Figure 8 Higher or comparable DNA yields observed when reverse crosslinking is done overnight at 80°C. Average yield (ng/lOum FFPE coupe) is plotted for the recommended procedure of the commercial kits [A-C] or short incubation of lhr at 80 °C [D] (left bar) and 16 hours at 80 °C [A-D] (right bar). The fold difference is shown underneath each graph. Error bars represent SEM.
  • Figure 9 FFPE DNA amplifiability improves when reverse crosslinking is done overnight at 80°C. Average number of amplifiable DNA molecules per lOpg FFPE DNA is plotted for both the recommended procedure of the commercial kits [A-C] or short incubation of lhr at 80 °C [D] (left bar) and 16 hours at 80 °C [A-D] (right bar). The calculated average is obtained from processing five independent clinical FFPE samples with the QPCR performed in triplicate . The fold difference is shown underneath each graph. Error bars represent SEM.
  • Figure 10 Average number of amplifiable DNA molecules per FFPE curl from five different samples is plotted for both the recommended procedure of the commercial kits [A-C] or short incubation of lhr at 80 °C [D] (left bar) and 16 hours at 80 °C [A-D] (right bar). The fold difference is shown underneath each graph. Error bars represent SEM.
  • the present methods comprise a step of reversing the cross-linking of a sample comprising cross-linked polynucleotides wherein the sample is incubated at about 75 °C to about 87 °C for about 8 hours to about 24 hours.
  • Reversing crosslinking is used herein to refer to breaking the crosslinks such that the DNA that has been crosslinked is no longer crosslinked and is suitable for subsequent amplification and/or sequencing steps.
  • the present methods may comprise incubating the sample at about 75 °C to about 87 °C.
  • the present methods may comprise incubating the sample at about 80 °C to about 87 °C.
  • the present methods may comprise incubating the sample at about 80 °C to about 85 °C.
  • the present methods may comprise incubating the sample at about 80 °C, about °C, about 82 °C, about 83 °C, about 84 °C, about 85 °C, about 86 °C, or about 87 °C.
  • the present methods may comprise incubating the sample for about 8 hours to about 24 hours.
  • the present methods may comprise incubating the sample for about 10 hours to about 18 hours.
  • the present methods may comprise incubating the sample for about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, or about 18 hours.
  • the present methods may comprise incubating the sample for about 16 hours.
  • the present methods may comprise incubating the sample at about 80 °C to about 87 °C for about 8 hours to about 24 hours.
  • the present methods may comprise incubating the sample at about 80 °C to about 87 °C for about 10 hours to about 18 hours.
  • the present methods may comprise incubating the sample at about 80 °C to about 87 °C for about 16 hours.
  • the present methods may comprise incubating the sample at about 80 °C to about 85 °C for about 10 hours to about 18 hours.
  • the present methods may comprise incubating the sample at about 80 °C to about 85 °C for about 16 hours.
  • the present methods may comprise incubating the sample at about 85 °C for about 10 hours to about 18 hours.
  • the present methods may comprise incubating the sample at about 85 °C for about 16 hours.
  • the sample may also be treated with a proteinase, for example proteinase K, in order to digest the protein.
  • a proteinase for example proteinase K
  • the proteinase treatment may be performed before and/or after the reverse crosslinking step of the invention.
  • Suitable conditions for performing a proteinase K treatment are known in the art, for example the sample may be incubated at 37°C to 65°C for 30 minutes to 16 hours, for example 1 to 4 hours.
  • the reverse cross-linking step does not comprise contacting the sample with an adduct reversal agent.
  • An “adduct reversal agent” as used herein may refer to an agent that reduces the number of adducts and/or cross-links in a sample comprising cross-linked polynucleotides.
  • an adduct reversal agent may be a compound that includes an amine group and a proton-donating group.
  • the amine group and the proton-donating group may be substituted on a cyclic group, such as an aromatic ring.
  • an adduct reversal agent is a compound that includes an aromatic ring and at least one of: an amine group and a proton-donating group.
  • an adduct reversal agent is a compound that includes an aromatic ring, an amino group and a proton-donating group.
  • an adduct reversal agent is selected from Aniline, 2-aminobenzoic acid, 2-amino-5-methoxybenzoic acid, 2 -amino-5 -methylphenyl phosphonic acid, 4-aminobenzoic acid, 3,5-diaminobenzoic acid, benzene-
  • the present reverse cross-linking step is performed in a buffer comprising a pH stabilising component which is capable of reacting with formaldehyde molecules released during the reverse cross- linking process.
  • a pH stabilising component which is capable of reacting with formaldehyde molecules released during the reverse cross- linking process.
  • suitable components include, but are not limited to, Tris-HCl or NaHCCE.
  • the buffer may comprise, for example, at least 10 mM, at least 20 mM, at least 40 mM, at least 60 mM or at least 100 mM Tris-HCl.
  • the buffer may comprise at least 10 mM Tris-HCl.
  • the present reverse cross-linking step is performed in a buffer comprising NaCl.
  • the presence of NaCl may be advantageous as it influences the temperature at which the DNA melts into two separate strands.
  • the buffer may comprise at least about 100 mM, at least about 150 mM, at least about 200 mM, at least about 400 mM, at least about 800 mM or at least about 500 mM NaCl.
  • the buffer may comprise at least about 200 mM NaCl.
  • the present reverse cross-linking step is performed in a buffer comprising a detergent.
  • the detergent may be sodium dodecyl sulfate (SDS).
  • the buffer may comprise at least 0.1%, at least 0.2%. at least 0.5%, at least 1%, at least 5% SDS.
  • the buffer may comprise at least 0.1% SDS.
  • a buffer comprising a pH stabilising component such as Tris-HCl or NaHCCE , NaCl and a detergent (such as SDS) may improve the stability of the DNA during the reverse cross-linking and thus enable higher temperatures and/or longer time periods to be used during the reverse cross-linking whilst retaining the integrity of the DNA for downstream molecular analysis.
  • a pH stabilising component such as Tris-HCl or NaHCCE , NaCl and a detergent (such as SDS) may improve the stability of the DNA during the reverse cross-linking and thus enable higher temperatures and/or longer time periods to be used during the reverse cross-linking whilst retaining the integrity of the DNA for downstream molecular analysis.
  • the buffer may comprise guanidinium thiocyanate.
  • Exemplary, non-limiting buffers in which the reverse cross-linking step may be performed are as follows. It is to be noted that these buffers are provided as non-exhaustive examples.
  • the buffer may comprise 1% SDS, 100 mM NaHCCE, and 200 mM NaCl; or 100 mM NaCl, lOmM Tris-HCl pH8, 25 mM EDTA pH8 and 0.5% SDS; or 66 mM Tris, 0.3% SDS, 1.3% TX-100 or Ecosurf-SA9, 443 mM NaCl, 21 mM potassium acetate, 4 mM Magnesium acetate, 4 mM DTT, 4 mM MgCh.
  • the reverse cross-linking of the present methods is sufficient to dissociate essentially all protein-DNA interactions within the sample. Accordingly, the reverse cross-linking step of the present methods is typically not suitable to be performed upstream of a chromatin-immunoprecipitation.
  • the present reverse cross-linking step is performed at the standard pressure which occurs when incubating the sample for the time and temperature as defined herein; for example in a standard laboratory screw cap or push-top 1.5ml Eppendorf tube.
  • the sample is not subject to any additional means of increasing the pressure under which the incubation is performed.
  • the present reverse cross-linking step is performed at atmospheric pressure.
  • the present reverse cross-linking step is performed at less than 500, 300, 200, 100, 50, or 20 psi.
  • a sample of cross-linked polynucleotides comprises a sample comprising polynucleotides which has been subjected to cross-linking.
  • Samples may be taken from a patient and/or from diseases tissue, and may also be derived from other organisms or from separate sections of the same organism, such as samples from one patient, one sample from healthy tissue and one sample from diseased tissue. Samples may thus be analysed according to the invention and compared with a reference sample, or different samples may be analysed and compared with each other. For example, from a patient being suspected of having breast cancer, a biopsy may be obtained from the suspected tumour. Another biopsy may be obtained from non-diseased tissue. From both tissue biopsies may be analysed according to the invention.
  • Genomic regions of interests may be the BRCA1 and BRCA2 gene, which genes are 83 and 86 kb long.
  • the present methods are for use with samples that have undergone a heavy cross-linking procedure (e.g. a fixation procedure).
  • a heavy cross-linking procedure e.g. a fixation procedure
  • suitable cross-linked samples include, but are not limited to, sample cross-linked with formalin and formaldehyde.
  • the sample is a formalin cross-linking sample.
  • the sample may be a paraffin embedded sample.
  • the sample may be a Formalin-Fixed Paraffin-Embedded (FFPE) sample.
  • the sample may be a tissue sample.
  • the sample may be a tumour sample.
  • the sample may be a FFPE tumour sample.
  • the sample may be a slice or a puncture from a FFPE sample.
  • the cross-linked polynucleotides may be cross-linked DNA.
  • the cross-linked DNA may be part of a chromatin complex.
  • sample of crosslinked DNA is a sample DNA which has been subjected to crosslinking.
  • Crosslinking the sample DNA has the effect that the three-dimensional state of the DNA within the sample remains largely intact. This way, DNA strands that are in physical proximity of each other remain in each others vicinity.
  • PARAFFIN REMOVAF Embodiments of the present methods in which the sample is paraffin embedded, for example an FFPE sample, may comprise an initial paraffin removal step.
  • Suitable methods for paraffin removal include, for example, xylene treatment, sonication and/or boiling the sample for a short time period (e.g. at least 80°C for around 3 minutes).
  • the present methods may be applied in circumstances in which it is required to reverse the cross-linking of polynucleotides and/or extract cross-linked polynucleotides for downstream analysis such as PCR- based techniques or sequencing.
  • the polynucleotide to be extracted may be DNA and/or RNA.
  • the present methods may be applied to extract and/or reverse the crosslinking of cross-linked polynucleotides from a sample for downstream use in DNA or RNA PCR and/or sequencing applications.
  • the present invention provides methods of determining at least part of the sequences of DNA fragments and methods of determining the presence or absence of a mutation in a genomic region of interest from a sample of fragmented cross-linked DNA; which methods comprise the step of reversing the cross- linking of the sample comprising cross-linked polynucleotides by incubating the sample at about 75 °C to about 87 °C for about 8 hours to about 24 hours.
  • genomic region of interest is a DNA sequence of an organism of which it is desirable to determine, at least part of, the DNA sequence.
  • a genomic region which is suspected of comprising an allele associated with a disease may be a genomic region of interest.
  • allele(s) means any of one or more alternative forms of a gene at a particular locus.
  • loci plural locus on a chromosome.
  • One allele is present on each chromosome of the pair of homologous chromosomes.
  • two alleles and thus two separate (different) genomic regions of interest may exist.
  • a “nucleic acid” or “polynucleotide” as referred to herein may include any polymer or oligomer of pyrimidine and purine bases, preferably cytosine, thymine, and uracil, and adenine and guanine, respectively.
  • the present invention contemplates any deoxyribonucleotide, ribonucleotide or peptide nucleic acid component, and any chemical variants thereof, such as methylated, hydroxymethylated or glycosylated forms of these bases, and the like.
  • the polymers or oligomers may be heterogeneous or homogenous in composition, and may be isolated from naturally occurring sources or may be artificially or synthetically produced.
  • the nucleic acids may be DNA or RNA, or a mixture thereof, and may exist permanently or transitionally in single -stranded or double -stranded form, including homoduplex, heteroduplex, and hybrid states.
  • identifier is a short sequence that can be added to an adaptor or a primer or included in its sequence or otherwise used as label to provide a unique identifier.
  • sequence identifier or tag
  • can be a unique base sequence of varying but defined length, typically from 4-16 bp used for identifying a specific nucleic acid sample. For instance 4 bp tags allow 4(exp4) 256 different tags. Typical examples are ZIP sequences, known in the art as commonly used tags for unique detection by hybridization. Identifiers are useful according to the invention, as by using such an identifier, the origin of a (PCR) sample can be determined upon further processing.
  • the different nucleic acid samples may be identified using different identifiers. For instance, as according to the invention sequencing may be performed using high throughput sequencing, multiple samples may be combined. Identifiers may then assist in identifying the sequences corresponding to the different samples. Identifiers may also be included in adaptors for ligation to DNA fragments assisting in DNA fragment sequences identification. Identifiers preferably differ from each other by at least two base pairs and preferably do not contain two identical consecutive bases to prevent misreads. The identifier function can sometimes be combined with other functionalities such as adaptors or primers.
  • aligning and “alignment” is meant the comparison of two or more nucleotide sequence based on the presence of short or long stretches of identical or similar nucleotides. Methods and computer programs for alignment are well known in the art.
  • “Fragmenting” includes any technique that, when applied to polynucleotides, in particular DNA which may be crosslinked DNA or not, results in fragments. Techniques well known in the art are sonication, shearing and/or enzymatic restriction, but other techniques can also be envisaged.
  • DNA is cross-linked during the preparation of FFPE samples, in particular because of the formalin fixation performed. Further, the preparation of FFPE samples results in fragmentation of the DNA.
  • the DNA extracted from an FFPE sample may have an average fragment size of around 400 bases, around 700 bases, around 1500 bases, around 3000 bases, around 6000 bases or around 10000 bases.
  • further fragmentation steps of the present methods may be optional.
  • the sample is an FFPE sample and further fragmentation step may be optional.
  • the DNA fragments that originate from a genomic region of interest remain in proximity of each other because they are crosslinked.
  • DNA fragments of the genomic region of interest which are in the proximity of each other due to the crosslinks, are ligated.
  • This type of ligation may also be referred to as proximity ligation.
  • DNA fragments comprising the target nucleotide sequence may ligate with DNA fragments within a large linear distance on sequence level.
  • sequences of DNA fragments within the spatial surrounding of the genomic region of interest are obtained.
  • Each individual target nucleotide sequence is likely to be crosslinked to multiple other DNA fragments.
  • often more than one DNA fragment may be ligated to a fragment comprising the target nucleotide sequence.
  • a sequence of the genomic region of interest may be built.
  • a DNA fragment ligated with the fragment comprising the target nucleotide sequence includes any fragment which may be present in ligated DNA fragments.
  • the sample for use in the present methods comprises fragmented polynucleotides, in particular fragmented DNA.
  • samples of crosslinked DNA may be further fragmented in step b) of embodiments of the present invention.
  • the fragmenting step b) may comprise sonication, and may be followed by enzymatic DNA end repair. Sonication results in the fragmenting of DNA at random sites, which can be either blunt ended, or can have 3’- or 5’- overhangs, as these DNA breakage points occur randomly, the DNA may be repaired (enzymatically), filling in possible 3’- or 5 ’-overhangs, such that DNA fragments are obtained which have blunt ends that allow ligation of the fragments to adaptors and/or to each other in the subsequent step c). Alternatively, the overhangs may also be made blunt ended by removing overhanging nucleotides, using e.g. exonucleases.
  • the fragmenting step b) may comprise fragmenting with one or more restriction enzymes, or combinations thereof. Fragmenting with a restriction enzyme is advantageous as it may allow control of the average fragment size.
  • the fragments that are formed may have compatible overhangs or blunt ends that allow ligation of the fragments in the subsequent step c).
  • the fragmenting step b) may be performed using SI nuclease to generate blunt ended fragments.
  • the fragmenting step b) may be performed using DNasel.
  • for each subsample restriction enzymes with different recognition sites may be used.
  • a “restriction endonuclease” or “restriction enzyme” is an enzyme that recognizes a specific nucleotide sequence (recognition site) in a double -stranded DNA molecule, and will cleave both strands of the DNA molecule at or near every recognition site, leaving a blunt or a 3’- or 5 ’-overhanging end.
  • the specific nucleotide sequence which is recognized may determine the frequency of cleaving, e.g. a nucleotide sequence of 6 nucleotides occurs on average every 4096 nucleotides, whereas a nucleotide sequence of 4 nucleotides occurs much more frequently, on average every 256 nucleotides.
  • the present methods may comprise a “ligation” step.
  • “Ligating” involves the joining of separate DNA fragments.
  • the DNA fragments may be blunt ended, or may have compatible overhangs (sticky overhangs) such that the overhangs can hybridise with each other.
  • the joining of the DNA fragments may be enzymatic, with a ligase enzyme, DNA ligase.
  • a non-enzymatic ligation may also be used, as long as DNA fragments are joined, i.e. forming a covalent bond.
  • a phosphodiester bond between the hydroxyl and phosphate group of the separate strands is formed.
  • a fragment comprising a target nucleotide sequence may be crosslinked to multiple other DNA fragments, more than one DNA fragment may be ligated to the fragment comprising the target nucleotide sequence. This may result in combinations of DNA fragments which are in proximity of each other as they are held together by the cross links. Different combinations and/or order of the DNA fragments in ligated DNA fragments may be formed.
  • the recognition site of the restriction enzyme is known, which makes it possible to identify the fragments as remains of or reconstituted restriction enzyme recognition sites may indicate the separation between different DNA fragments.
  • the ligation step may be performed in the presence of an adaptor, ligating adaptor sequences in between fragments.
  • the adaptor may be ligated in a separate step. This is advantageous because the different fragments can be easily identified by identifying the adaptor sequences which are located in between the fragments.
  • the ligated DNA fragments may optionally be further fragmented, preferably with a restriction enzyme.
  • the optional first and second fragmenting step may be aimed at obtaining ligated DNA fragments of a size which is compatible with the subsequent amplification step and/or sequence determination step.
  • a second fragmenting step preferably with an enzyme may result in ligated fragment ends which are compatible with the optional ligation of an adaptor.
  • the second fragmenting step may be performed after reversing the crosslinking, however, it is also possible to perform the second fragmenting step and/or ligation step while the DNA fragments are still crosslinked.
  • the restriction enzyme recognition site of the second fragmentation step is longer than the recognition site of the restriction enzyme used in the first fragmentation step.
  • the second enzyme thus cuts at a lower frequency than the first enzyme. This means that the average DNA fragment size after the first fragmentation is smaller than the average fragment size obtained after the second fragmentation step. This way, in the first fragmenting step, relatively small fragments are formed, which are subsequently ligated. As the second restriction enzyme cuts less frequently, most of the DNA fragments may not comprise the restriction recognition site of the second restriction enzyme. Thus, when the ligated DNA fragments are subsequently fragmented in the second fragmentation step, many of the initial DNA fragments may remain intact.
  • the first optional fragmenting step is less frequent than the second optional fragmenting step, the result would be that the initial fragment are generally further fragmented, which may result in the loss of relatively large DNA sequences that are useful for building a contig.
  • the first optional fragmenting step is more frequent as compared to the second optional fragmenting step, such that DNA fragments may largely remain intact, i.e. are largely not further fragmented in the second optional fragmentation step.
  • An “adaptor” is a short double -stranded oligonucleotide molecule with a limited number of base pairs, e.g. about 10 to about 30 base pairs in length, which are designed such that they can be ligated to the ends of fragments.
  • Adaptors are generally composed of two synthetic oligonucleotides which have nucleotide sequences which are partially complementary to each other. When mixing the two synthetic oligonucleotides in solution under appropriate conditions, they will anneal to each other forming a double -stranded structure.
  • one end of the adaptor molecule may be designed such that it is compatible with the end of a restriction fragment and can be ligated thereto; the other end of the adaptor can be designed so that it cannot be ligated, but this does need not to be the case, for instance when an adaptor is to be ligated in between DNA fragments.
  • At least one adaptor is optionally ligated to the ligated DNA fragments.
  • the ends of the ligated DNA fragments need to be compatible with ligation of such an adaptor.
  • the ligated DNA fragments may be linear DNA
  • ligation of an adaptor may provide for a primer hybridisation sequence.
  • the adaptor sequence ligated with ligated DNA fragments comprising the target nucleotide sequence provides DNA molecules which may be amplified using PCR.
  • DNA comprising the target nucleotide sequence may be amplified using at least one oligonucleotide primer which hybridises to the target nucleotide sequence, and at least one additional primer which hybridises to the at least one adaptor.
  • the DNA comprising the target nucleotide may also be amplified in using at least one oligonucleotide primer which hybridises to the target nucleotide sequence.
  • “Amplifying” refers to a polynucleotide amplification reaction, namely, a population of polynucleotides that are replicated from one or more starting sequences. Amplifying may refer to a variety of amplification reactions, including but not limited to polymerase chain reaction (PCR), linear polymerase reactions, nucleic acid sequence- based amplification, rolling circle amplification and like reactions.
  • PCR polymerase chain reaction
  • linear polymerase reactions nucleic acid sequence- based amplification
  • rolling circle amplification rolling circle amplification and like reactions.
  • Oligonucleotide primers in general, refer to strands of nucleotides which can prime the synthesis of DNA. DNA polymerase cannot synthesize DNA de novo without primers. A primer hybridises to the DNA, i.e. base pairs are formed. Nucleotides that can form base pairs, that are complementary to one another, are e.g. cytosine and guanine, thymine and adenine, adenine and uracil, guanine and uracil. The complementarity between the primer and the existing DNA strand does not have to be 100%, i.e. not all bases of a primer need to base pair with the existing DNA strand.
  • nucleotides are incorporated using the existing strand as a template (template directed DNA synthesis).
  • template directed DNA synthesis we may refer to the synthetic oligonucleotide molecules which are used in an amplification reaction as “primers”.
  • “Sequencing” refers to determining the order of nucleotides (base sequences) in a nucleic acid sample, e.g. DNA or RNA. Many techniques are available such as Sanger sequencing and High throughput sequencing technologies such as offered by Roche, Illumina and Applied Biosystems.
  • the sequence of the (amplified) ligated DNA fragments for example comprising the target nucleotide sequence is determined. Determining the sequence is preferably performed using high throughput sequencing technology, as this is more convenient and allows a high number of sequences to be determined to cover the complete genomic region of interest. From these determined sequences a contig may be built of the genomic region of interest. When sequences of the DNA fragments are determined, overlapping reads may be obtained from which the genomic region of interest may be built. In case the DNA fragments were obtained by random fragmentation, the random nature of the fragmentation step already may result in DNA fragments which when sequenced results in overlapping reads. By increasing the sample size, e.g. increasing the number of cells analysed, the reliability of the genomic region of interest that is built may be increased.
  • sequencing adaptors may be ligated to the (amplified) ligated DNA fragments.
  • the linear or circularized fragment is amplified, by using for example PCR as described herein, the amplified product is linear, allowing the ligation of the adaptors.
  • Suitable ends may be provided for ligating adaptor sequences (e.g. blunt, complementary staggered ends).
  • primer(s) used for PCR or other amplification method may include adaptor sequences, such that amplified products with adaptor sequences are formed in the amplification.
  • the circularized fragment may be fragmented, preferably by using for example a restriction enzyme in between primer binding sites for the inverse PCR reaction, such that DNA fragments ligated with the DNA fragment comprising the target nucleotide sequence remain intact.
  • Sequencing adaptors may also be included in the ligation steps of the methods of the invention. These sequencing adaptors may be included as part of the adaptor sequences of the adaptors that may already optionally used in these steps and/or separate sequence adaptors may be provided in these steps in addition.
  • long reads may be generated in the high throughput sequencing method used.
  • Long reads may allow one to read across multiple DNA fragments of ligated DNA fragments. This way, initial DNA fragments or DNA fragments generated during optional further fragmentation in step (b) may be identified.
  • DNA fragment sequences may be compared to a reference sequence and/or compared with each other. For example, such DNA fragment sequences may be used for determining the ratio of fragments of cells carrying a genetic mutation.
  • sequencing also DNA fragment sequences of DNA fragments adjacent to such sequences, unique ligated DNA fragments may be identified. This is in particular the case when DNA fragments were obtained in step by random fragmentation.
  • Such short reads may involve additional processing steps such that separate ligated DNA fragments when fragmented, are ligated or equipped with identifiers, such that from the short reads, contigs may be built for the ligated DNA fragments.
  • Such high throughput sequencing technologies involving short sequence reads may involve paired end sequencing.
  • the short reads from both ends of a DNA molecule used for sequencing which DNA molecule may comprise different DNA fragments, may allow coupling of DNA fragments that were ligated. This is because two sequence reads can be coupled spanning a relatively large DNA sequence relative to the sequence that was determined from both ends. This way, contigs may be built for the (amplified) ligated DNA fragments.
  • short reads may be contemplated without identifying DNA fragments, because from the short sequence reads a genomic region of interest may be built, especially when the genomic region of interest has been amplified. Information regarding DNA fragments and/or separate genomic region of interests (for instance of a diploid cell) may be lost, but DNA mutations may still be identified.
  • the step of determining at least part of the sequence of the (amplified) ligated DNA sequence may comprise short sequence reads, but preferably longer sequence reads are determined such that DNA fragment sequences may be identified.
  • a contig is used in connection with DNA sequence analysis, and refers to reassembled contiguous stretches of DNA derived from two or more DNA fragments, preferably three or more DNA fragments, having contiguous nucleotide sequences.
  • a contig may be a set of overlapping DNA fragments that provides a (partial) contiguous sequence of a genomic region of interest.
  • a contig may also be a set of DNA fragments that, when aligned to a reference sequence, may form a contiguous nucleotide sequence.
  • the term "contig” encompasses a series of (ligated) DNA fragment(s) which are ordered in such a way as to have sequence overlap of each (ligated) DNA fragment(s) with at least one of its neighbours.
  • the linked or coupled (ligated) DNA fragment(s) may be ordered either manually or, preferably, using appropriate computer programs such as FPC, PHRAP, CAP3 etc, and may also be grouped into separate contigs.
  • the present methods may optionally comprise a step of circularization the DNA fragments generated, for example following reverse cross-linking.
  • a restriction enzyme is used for the second fragmentation, as a restriction enzyme allows control of the fragmentation step and results, if an appropriate restriction enzyme is chosen, in compatible ends of the ligated DNA fragments that are favourable for ligation of the compatible ends, resulting in circularized ligated DNA fragments.
  • fragmenting using other methods e.g. shearing and/or sonication and subsequent enzymatic DNA end repair, such that blunt ended double strand DNA is formed may also be ligated to form circularized DNA.
  • the optional first and second fragmenting steps are aimed at obtaining ligated DNA fragments which are compatible with the subsequent circularization, amplification step and/or sequence determination step.
  • the optional first and second fragmenting steps comprise restriction enzymes
  • the first and second restriction enzyme may be chosen as described herein.
  • Circularization involves the ligation of the ends of the ligated DNA fragments such that a closed circle is formed.
  • the circularized DNA comprising ligated DNA fragments which comprise the target nucleotide sequence may subsequently be amplified using at least one primer which hybridises to the target nucleotide sequence.
  • amplification step reversing the crosslinking is required, as crosslinked DNA may hamper or prevent amplification.
  • two primers are used that hybridise to the target nucleotide sequence in an inverse PCR reaction. In this way, DNA fragments of the circularized DNA, which are ligated with the DNA fragment comprising the target nucleotide sequence, may be amplified.
  • Size selection involves techniques with which particular size ranges of molecules, e.g. (ligated) DNA fragments or amplified (ligated) DNA fragments, are selected. Techniques that can be used are for instance gel electrophoresis, size exclusion, gel extraction chromatography, but are not limited thereto, as long as molecules with a particular size can be selected, such a technique will suffice.
  • a size selection step may be performed prior to or after the amplification.
  • a size selection step may be performed using gel extraction chromatography, gel electrophoresis or density gradient centrifugation, which are methods generally known in the art.
  • DNA is selected of a size between 20-20,0000 base pairs, preferably 50-10,0000 base pairs, most preferably between 100-3,000 base pairs.
  • a size separation step allows to select for (amplified) ligated DNA fragments in a size range that may be optimal for PCR amplification and/or optimal for the sequencing of long reads by next generation sequencing.
  • SMRTTM Single Molecule Real Time
  • PLOIDY In case the ploidy in a cell of a genomic region of interest is greater than 1, for each ploidy a sequence may be determined, the presence or absence of a mutation determined and/or a contig may be built.
  • genomic environment of any given target site in the genome mostly consists of DNA genome sequences that are physically close to the target sequence on the linear chromosome template, it allows the reconstruction of each particular chromosome template.
  • ploidy of a genomic region of interest is greater than 1, multiple genomic regions of interest are present in a cell (or equivalent thereof). These multiple genomic regions of interest generally do not occupy the same space, i.e. they are separated in space.
  • a sample of crosslinked DNA of such a cell is fragmented, from each genomic region of interest in a cell a corresponding DNA fragment comprising the target nucleotide sequence will be formed. These DNA fragments will each ligate with DNA fragments in their proximity. Ligated DNA fragments will thus be representative of the different genomic regions of interest.
  • the step determining the sequence of at least part of the sequences of DNA fragments, determining the presence or absence of a mutation or of building a contig comprises the steps of:
  • the step 2) of assigning the fragments to a genomic region comprises identifying the different ligation products and coupling of the different ligation products comprising the DNA fragments.
  • the methods of the present invention may be used for haplotyping.
  • heterogeneous cell populations e.g. cells with different origin or cells from an organism which comprises normal cells and genetically mutated cells (e.g. cancer cells)
  • a sequence may be determined, the presence or absence of a mutation determined and/or a contig may be built.
  • Methods are provided for identifying the presence or absence of a genetic mutation.
  • the method for identifying the presence or absence of a genetic mutation may comprise the steps of any of methods of the invention as described above and further identifying the presence or absence of a genetic mutation in a sequence determined.
  • Genetic mutations can be identified for instance by sequences determined for multiple samples, in case one (or more) of the samples comprises a genetic mutation, this may be observed as the sequence is different when compared to the sequence of the other samples, i.e. the presence of a genetic mutation is identified. In case no sequence differences between the samples is observed, the absence of genetic mutation is identified.
  • a reference sequence may also be used to which the sequence may be aligned. When the sequence of the sample is different from the sequence of the reference sequence, a genetic mutation is observed, i.e. the presence of a genetic mutation is identified. In case no sequence differences between the sample or samples and the reference sequence is observed, the absence of genetic mutation is identified.
  • DNA fragments sequences may be aligned, with each other or with a reference sequence, the presence or absence of a genetic mutation may be identified.
  • an identified genetic mutation may be a single nucleotide variant (SNV), for example a single nucleotide polymorphism (SNP), an insertion, an inversion and/or a translocation.
  • SNV single nucleotide variant
  • SNP single nucleotide polymorphism
  • the number of fragments and/or ligation products from a sample carrying the deletion and/or insertion may be compared with a reference sample in order to identify the deletion and/or insertion.
  • a deletion, insertion, inversion and/or translocation may also be identified based on the presence of chromosomal breakpoints in analyzed fragments.
  • the presence or absence of methylated nucleotides is determined in DNA fragments, ligated DNA fragments, and/or genomic regions of interest.
  • the DNA may be treated with bisulphite.
  • Treatment of DNA with bisulphite converts cytosine residues to uracil, but leaves 5-methylcytosine residues unaffected.
  • bisulphite treatment introduces specific changes in the DNA sequence that depend on the methylation status of individual cytosine residues, yielding single- nucleotide resolution information about the methylation status of a segment of DNA.
  • sequences from a plurality of samples treated with bisulphite may also be aligned, or a sequence from a sample treated with bisulphite may be aligned to a reference sequence.
  • the primer sequence may be removed prior to the high throughput sequencing step.
  • primers are used carrying a moiety, e.g. biotin, for the optional purification of (amplified) ligated DNA fragments through binding to a solid support.
  • a moiety e.g. biotin
  • the ligated DNA fragments comprising the target nucleotide sequence may be captured with a hybridisation probe (or capture probe) that hybridises to a target nucleotide sequence.
  • the hybridisation probe may be attached directly to a solid support, or may comprise a moiety, e.g. biotin, to allow binding to a solid support suitable for capturing biotin moieties (e.g. beads coated with streptavidin).
  • the ligated DNA fragments comprising a target nucleotide sequence are captured thus allowing to separate ligate DNA fragments comprising the target nucleotide sequence from ligated DNA fragments not comprising the target nucleotide sequence.
  • an amplification step is performed, which is also an enrichment step
  • a capture step with a probe directed to a target nucleotide sequence may be performed.
  • a genomic region of interest at least one capture probe for a target nucleotide sequence may be used for capturing.
  • more than one probe may be used for multiple target nucleotide sequences.
  • one primer of one of the 5 target nucleotide sequences may be used as a capture probe (A, B, C, D or E).
  • the 5 primers may be used in a combined fashion (A, B, C, D and E) capturing the genomic region of interest.
  • an amplification step and capture step may be combined, e.g. first performing a capture step and than an amplification step or vice versa.
  • a capture probe may be used that hybridises to an adaptor sequence comprised in (amplified) ligated DNA fragments.
  • This disclosure is not limited by the exemplary methods and materials disclosed herein, and any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of this disclosure. Numeric ranges are inclusive of the numbers defining the range. Unless otherwise indicated, any nucleic acid sequences are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively.
  • Figure 1A shows an agarose gel image of the purified DNA. Arrows in Figure IB point at DNA in the slots (65-80 °C) which is likely to represent incomplete reverse crosslinked DNA molecules and of which the quantity declines with an increase in temperature. The arrow at 90°C points towards DNA that is running at a much lower molecular weight than expected, likely explained by DNA fragmentation.
  • Figure 4 shows the fold change of amplifiability relative to the 16h, 65 °C condition of the seven different sized amplicons.
  • the relative fold change is larger for the longest amplicons from the 80 and 85°C conditions. This finding indicates that the reverse crosslinking occurs more effectively in these conditions but that there is low DNA damage. The longer fragments are more prone to DNA damage because of their size. At the 90°C and 95 °C conditions there is no relation between size and amplifiability . Combined with the observation that DNA yields improve under these conditions this can be explained by a more complete reverse crosslinking taking place, but either DNA degradation takes place or reversal is not sufficiently complete to allow effective PCR amplification.
  • the 24 h 80°C condition yields a 2.5 to 7.6 fold increase in amplifiable products as can be seen in Figure 4.
  • the highest increase is in the largest product as seen above.
  • FFPE DNA from different tissue blocks was extracted after proteinase K treatment and the various reverse crosslinking treatments using magnetic beads and isopropanol precipitation. Each sample was eluted in the same volume and individually measured with Qubit.
  • Figure 1A shows the DNA concentrations of the two different FFPE tissues that were used for this experiment.
  • Figure IB shows the size of the DNA on an 0.6% agarose gel. Equal amounts of DNA were loaded in each lane.
  • Amplifiability of reverse crosslinked DNA was initially tested using an end-point PCR reaction.
  • a lOplex-multiplex end-point PCR is performed on FFPE samples isolated after overnight reverse crosslinking at temperatures ranging from 65 to 90°C. PCR reactions are analyzed on a 2% gel shown in Figure 2.
  • Reverse crosslinking conditions tested a variety of treatments and comparing this to a standard treatment of 16hour at 65°C. For comparison two standard treatments used by commercial suppliers (lh at 90 and 80°C) were included. FFPE DNA from different tissue blocks was extracted after proteinase K treatment and the various heat treatments using magnetic beads and isopropanol precipitation. Each sample was eluted in the same volume and individually measured with Qubit. Figure 3 shows the relative yield for the three different FFPE tissues compared to the treatment of 16h at 65°C. Commercial DNA isolation procedures typically use shorter incubations and sometimes higher temperatures (e.g.
  • Results described in Figure 4 and 5 were obtained using sample 961, the sample showing no improved DNA yields following the higher reverse crosslinking temperature incubations. Next the QPCR assay was applied on all three FFPE samples using three different sized amplicons.
  • Figure 6 shows the fold change of the three amplicons in different FFPE tissues at different conditions. All three tissues show the benefit of the 80-85°C conditions versus the standard 16h 65°C, again with the largest benefit for the longest amplicons. Shorter incubations ( ⁇ 4hrs) at temperatures >90°C does not result in improved amplifiability.
  • Kit B includes an incubation buffer comprising guanidinium thiocyanate.
  • the quantity of DNA isolated from an FFPE sample does not necessarily directly correlate with the quality of the DNA molecules.
  • the KAPA Human Genomic DNA Quantification Kit (Roche) in triplicate allowing us to calculate the number of amplifiable DNA molecules of three different sized amplicons (41, 129 and 305bp) starting from lOpg of FFPE DNA isolated from the different samples as determined by Qubit. Per amplicon and per condition the average number of amplifiable molecules was calculated and depicted in Figure 9.
  • dsDNA breaks often observed in FFPE samples will not be repaired with the longer incubation time, which likely explains the amplifiability of the longer amplicons is reduced when compared to the shorter amplicons.
  • the increase in total number of amplifiable molecules per sample is important, as FFPE samples are known to show lower performance in certain assays compared to DNA isolated from fresh samples. The more molecules per sample can be isolated; the more information can be retrieved so the more reliable the outcome of the assay will be.

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Abstract

La présente invention concerne un procédé d'inversion de la réticulation d'un échantillon comprenant des polynucléotides réticulés, le procédé comprenant l'incubation de l'échantillon à une température comprise entre environ 75 °C et environ 87 °C pendant une durée comprise entre environ 8 heures et environ 24 heures.
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Citations (2)

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WO2010065775A2 (fr) * 2008-12-03 2010-06-10 The United States Government As Represented By The Department Of Veterans Affairs Récupération moléculaire assistée par pression (pamr) de biomolécules, extraction d'antigène assistée par pression (paar) et histologie de tissu assistée par pression (path)
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