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WO2025151541A1 - Extraction d'adn génomique à partir de cellules fixes - Google Patents

Extraction d'adn génomique à partir de cellules fixes

Info

Publication number
WO2025151541A1
WO2025151541A1 PCT/US2025/010783 US2025010783W WO2025151541A1 WO 2025151541 A1 WO2025151541 A1 WO 2025151541A1 US 2025010783 W US2025010783 W US 2025010783W WO 2025151541 A1 WO2025151541 A1 WO 2025151541A1
Authority
WO
WIPO (PCT)
Prior art keywords
cells
dna
salt
linking
fixed
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/US2025/010783
Other languages
English (en)
Inventor
Jason I. COMANDER
Kannan Vrindavan MANIAN
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Massachusetts Eye and Ear
Original Assignee
Massachusetts Eye and Ear Infirmary
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Massachusetts Eye and Ear Infirmary filed Critical Massachusetts Eye and Ear Infirmary
Publication of WO2025151541A1 publication Critical patent/WO2025151541A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay

Definitions

  • This invention relates to purification of nucleic acids.
  • the cells of a) are fixed in paraformaldehyde (PFA), formaldehyde, glutaraldehyde, formalin, 10% neutral buffered formalin (NBF)), glyoxal, or acrolein.
  • PFA paraformaldehyde
  • formaldehyde glutaraldehyde
  • formalin 10% neutral buffered formalin (NBF)
  • NBF neutral buffered formalin
  • DNA obtained from step c) is further purified.
  • DNA purification includes using a silica-based column.
  • recovered DNA is genomic DNA.
  • genomic DNA precipitated in step d is of high molecular weight of at least 20 kilo base-pairs.
  • the cells are fixed in a biological sample embedded in an embedding medium. In some embodiments, the embedding material has been removed.
  • the biological sample is a tissue. In some embodiments, the biological sample is on a support (e.g., a glass slide). In any of the aforementioned embodiments, the cells or biological samples (e.g., a tissue sample) are human cells or human biological samples.
  • the cells or biological samples are mammalian cells (e.g., mouse, rat, guinea pig, hamster, dog, rabbit, monkey or cells or biological samples, cells, or tissues of a laboratory animal).
  • mammalian cells e.g., mouse, rat, guinea pig, hamster, dog, rabbit, monkey or cells or biological samples, cells, or tissues of a laboratory animal.
  • Figures 1 A-1C show representative images of genomic DNA characterization extraction from a range (2-8%) of paraformaldehyde-fixed cells.
  • Figure 1 A shows phase separation after the addition of isopropanol.
  • Figure 1 B shows high molecular weight genomic DNA (1 pg) from cells fixed with different PFA concentrations, resolved on 1% agarose gel.
  • Figure 1C shows a polymerase chain reaction (PCR) of the same DNA amplifying 1.2 kb rhodopsin transgene and ⁇ 10 kb beta-globin from 2%, 4%, and 8% PFA fixed cells (range of concentration tested).
  • PCR polymerase chain reaction
  • Cross-linking fixation of mammalian cells can facilitate cell-based assays like immunostaining and fluorescence-activated cell sorting (FACS).
  • FACS fluorescence-activated cell sorting
  • high-quality genomic DNA from fixed mammalian cells cannot generally be obtained using traditional DNA isolation protocols due cross-linked protein-DNA complexes and DNA fragmentation.
  • PFA paraformaldehyde
  • the protocol extracts DNA from PFA fixed mammalian cells.
  • the protocol describes reverse crosslinking using heat, treatment with a high salt concentration, RNase A treatment, followed by isopropanol precipitation, and further DNA purification using a silica-based column.
  • This protocol yields significantly high-quality unfragmented DNA, which is PCR-amplifiable DNA, and gives better downstream results than without crosslinking reversal, especially for high throughput NGS analysis.
  • a combination of a low salt lysis buffer and high salt intermediate step followed by precipitation was found to retain both DNA integrity and yield from fixed cells.
  • a 5M NaCI (sodium chloride) addition and incubation on ice for 10 minutes was found to further improve yield of genomic DNA.
  • moderate temperatures ranging from 55-70°C and preferably 60°C preserves DNA integrity.
  • fixed cells are provided and used in the methods disclosed herein.
  • Such cells having been fixed with, for example, an aldehyde, for example, with paraformaldehyde, formaldehyde, formalin, 10% neutral buffered formalin, or glutaraldehyde, glyoxal, acrolein or other known fixatives.
  • Cell fixation methods are known in the art including, for example, cells having been fixed for 20 minutes at room temperature with 4% paraformaldehyde. Standard methods for fixing cells are described, for example, in Jamur et al., Methods Mol Biol, 2010. 588: p. 55-61 and Banerjee et al. Biotechniques, 2018. 65(2): p. 65- 69.
  • isolating high quality DNA from fixed cells involves employing moderate temperature decrosslinking, a high salt intermediate step, and avoiding shearing the DNA.
  • a biological sample such as a cell or tissue sample
  • the fixed sample is processed, for example, by dehydrating, clearing, and/or embedding the fixed sample.
  • the fixed (and optionally processed sample) is then used to recover genomic DNA as disclosed herein. If the tissue is embedded in a matrix, then such matrix is removed and dissolved in a separate step (e.g., by employing xylene to remove paraffin) according to standard methods.
  • tissue samples that can be used in the methods disclosed herein include, but are not limited to, whole organs or a portion thereof, organ sub-structures, tissue biopsies, punch biopsies, fine-needle aspirate biopsies, bone, archival tissues, or cells.
  • these biological samples are referred to as “tissues” or “tissue samples.” In cases where a sample is large, it can be cut into smaller pieces (such as pieces 1-3 mm thick or less), for ease of handling and improved fixative penetration and processing.
  • biological samples, cells, tissues may be processed according to standard histological methods, for example, by adhering to a support such as a glass slide.
  • Cells may, for example, be smeared on slides, fixed, and processed for recovery of DNA as described.
  • Biological samples on glass slides may be gently shaken during processing.
  • DNA is extracted from the tissue samples (e.g., an embedded sample) or sections and utilized for analyses such as PCR, real-time PCR, quantitative real-time PCR, microarray analysis, sequencing (e.g., next generation sequencing), and Southern blotting.
  • analyses such as PCR, real-time PCR, quantitative real-time PCR, microarray analysis, sequencing (e.g., next generation sequencing), and Southern blotting.
  • Reverse crosslinking of fixed cells is achieved according to standard methods.
  • Exemplary solutions employed at this step include salts for ionic strength (NaCI, KCI), salts for DNA stabilization or nuclease inhibition (MgCI2), a nuclease inhibitor (EDTA), a pH buffering agent (Tris-HCI), detergents (Nonidet P-40, Tween-20, Triton X-100, sodium dodecyl sulfate), and a protein blocking agent (gelatin, milk, serum, albumin).
  • an incubation with low salt containing buffer at temperature between 50-70°C, avoiding high temperatures such as 90-100°C, has been found to effectively reverse cross-links, which may be increased in the presence of proteinase K.
  • reverse cross-linking of fixed cells is achieved, for example, by agitation at a temperature of 60°C in the presence of a low salt lysis buffer and Proteinase K.
  • the reverse crosslinking step involves utilizing a low salt buffer for cell lysis and reverse crosslinking at 60°C.
  • a low salt buffer is the PDNB buffer described herein.
  • Such low salt buffer concentrations range from 10mM to 100 mM of a salt.
  • KCI is employed, for example at 50 mM.
  • a high concentration of salt is not preferred and is distinguished from adding an additional high salt step as described below.
  • RNAase A is added as is described here.
  • RNases such as RNase A in combination with RNase T1 (RNase cocktail enzyme mix, Thermo) may also be used.
  • DNA may be purified further as needed.
  • phases separate, and the bottom layer is enriched with precipitated DNA and collected, while the top aqueous layer (organic phase) lacked detectable DNA.
  • partially purified DNA may be exposed to an additional purification step on a column-contained matrix (e.g., a column with a silica- based binding matrix).
  • a column-contained matrix e.g., a column with a silica- based binding matrix.
  • This is a purification technique in which the DNA binds to the matrix (silica) with the help of chaotropic salts (guanidinium thiocyanate) in the buffer.
  • chaotropic salts guanidinium thiocyanate
  • 293T cells were transduced with a rhodopsin (RHO) expression cassette under the control of the EF1a promoter.
  • the cells were maintained in DMEM supplemented with 10% FBS.
  • FBS rhodopsin
  • 1 million cells were collected, pelleted, and fixed in 2 mL of 4% paraformaldehyde for 20 minutes at room temperature. After a PBS wash, the cell pellets were either stored at -80°C (long-term storage) or processed immediately for DNA extraction.
  • PDNB buffer contains 50 mM KCI, 10 mM Tris-HCI (pH 8.3), 2.5 mM MgCI2, 0.1 mg/ml gelatin, 0.45% (v/v) Nonidet P40, and 0.45% (v/v) Tween 20.
  • thermomixer set at 60°C at 600 r.p.m.
  • FIG. 1A Representative images of genomic DNA extracted from 2-4% paraformaldehyde-fixed cells are shown in Figures 1A-1C. Phase separation after the addition of isopropanol is shown in Fig. 1A. Genomic DNA (1 pg) from cells fixed with different PFA concentrations, resolved on 1% agarose gel is shown in Fig. 1b. PCR of the same DNA (shown in 1 B) amplifying 1.2 kb rhodopsin transgene and ⁇ 10 kb beta-globin from 2%, 4% and 8% PFA fixed cells (range of concentration tested) is shown in Fig. 1 C. Next, the amplicons from figure 1 B were subjected to next generation sequencing (NGS) to determine the intactness of the DNA sequence of the recovered DNA. Compared to the known original DNA sequence, the fixed DNA showed a very low sequence error rate, nearly equivalent to unfixed DNA, even without adding DNA modifying enzymes such as uracil N-glycosylase:
  • NGS next generation sequencing
  • EXAMPLE 4 Extraction of genomic DNA from cells embedded In a tissue
  • Murine retinas were subjected to the following fixation conditions for 20 minutes, and then exposed to the methods in Example 1.

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  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Health & Medical Sciences (AREA)
  • Biomedical Technology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Plant Pathology (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne un procédé comprenant les étapes suivantes : a) fourniture de cellules fixes réticulées ; b) réticulation inverse des cellules de l'étape a) ; c) incubation dans une solution à haute teneur en sel ; et d) précipitation de l'acide désoxyribonucléique (ADN) à partir des cellules. Le procédé est utile pour récupérer de l'ADN génomique de haute qualité.
PCT/US2025/010783 2024-01-08 2025-01-08 Extraction d'adn génomique à partir de cellules fixes Pending WO2025151541A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US202463618672P 2024-01-08 2024-01-08
US63/618,672 2024-01-08

Publications (1)

Publication Number Publication Date
WO2025151541A1 true WO2025151541A1 (fr) 2025-07-17

Family

ID=96387515

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2025/010783 Pending WO2025151541A1 (fr) 2024-01-08 2025-01-08 Extraction d'adn génomique à partir de cellules fixes

Country Status (1)

Country Link
WO (1) WO2025151541A1 (fr)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20170283860A1 (en) * 2014-09-16 2017-10-05 The Board Of Trustees Of The Leland Stanford Junio University Methods and compositions for the removal of aldehyde adducts and crosslinks from biomolecules
US20180237951A1 (en) * 2015-08-12 2018-08-23 Cemm - Forschungszentrum Für Molekulare Medizin Gmbh Methods for studying nucleic acids
US20210317506A1 (en) * 2018-05-08 2021-10-14 The University Of Chicago Chemical platform assisted proximity capture (cap-c)

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20170283860A1 (en) * 2014-09-16 2017-10-05 The Board Of Trustees Of The Leland Stanford Junio University Methods and compositions for the removal of aldehyde adducts and crosslinks from biomolecules
US20180237951A1 (en) * 2015-08-12 2018-08-23 Cemm - Forschungszentrum Für Molekulare Medizin Gmbh Methods for studying nucleic acids
US20210317506A1 (en) * 2018-05-08 2021-10-14 The University Of Chicago Chemical platform assisted proximity capture (cap-c)

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
KUEAKHAI PORNANAN, CHANGKLUNGMOA NARIN, CHAITHIRAYANON KULATHIDA, SONGKOOMKRONG SINEENART, RIENGROJPITAK SUDA, SOBHON PRASERT: "Production and characterization of a monoclonal antibody against recombinant saposin-like protein 2 of Fasciola gigantica", ACTA TROPICA, ELSEVIER, AMSTERDAM, NL, vol. 125, no. 2, 1 February 2013 (2013-02-01), AMSTERDAM, NL, pages 157 - 162, XP093337111, ISSN: 0001-706X, DOI: 10.1016/j.actatropica.2012.11.001 *

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