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WO2011026194A1 - Extraction d'acide nucléique - Google Patents

Extraction d'acide nucléique Download PDF

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Publication number
WO2011026194A1
WO2011026194A1 PCT/AU2010/001151 AU2010001151W WO2011026194A1 WO 2011026194 A1 WO2011026194 A1 WO 2011026194A1 AU 2010001151 W AU2010001151 W AU 2010001151W WO 2011026194 A1 WO2011026194 A1 WO 2011026194A1
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Prior art keywords
protease
nucleic acid
kit
cells
amplification
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PCT/AU2010/001151
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English (en)
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Christina Mccarthy
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Reproductive Health Science Pty Ltd
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Priority claimed from AU2009904275A external-priority patent/AU2009904275A0/en
Application filed by Reproductive Health Science Pty Ltd filed Critical Reproductive Health Science Pty Ltd
Publication of WO2011026194A1 publication Critical patent/WO2011026194A1/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
    • 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

  • the present invention relates generally to methods for extracting a nucleic acid from a sample comprising one or more cells containing the nucleic acid.
  • the present invention also contemplates amplification of the extracted nucleic acids, particularly whole genome amplification of the extracted nucleic acids.
  • WGA Whole Genome Amplification
  • WGA is used to amplify rare and/or low concentration genomic DNA templates, thus enabling a range of genotyping techniques that could not otherwise be performed.
  • WGA may be used to amplify genomic DNA derived from single cells for comparative genomic hybridisation.
  • the amplicons generated by WGA must be representative of the original genomic DNA template.
  • many WGA methodologies generate amplicons that have significant amplification bias and/or poorly represent or misrepresent the original genomic DNA. These problems limit the usefulness of WGA in methods such as comparative genomic hybridisation. Thus, it would be desirable to reduce amplification bias in WGA and/or to make WGA amplicons more representative of the original genomic DNA.
  • the present invention provides a method for extracting a nucleic acid from a sample comprising one or more cells containing the nucleic acid, the method comprising the steps of:
  • Digestion of one or more proteins associated with the nucleic acid is beneficial for further processing of the extracted nucleic acid.
  • digestion of one or more proteins associated with the nucleic acid may lead to higher quality amplicons being generated from amplification of the extracted nucleic acid and/or improved hybridisation of the extracted nucleic acid to a probe or primer.
  • the method of the present invention is adapted to the extraction of a nucleic acid from a single cell.
  • the present invention also provides a method for amplifying a nucleic acid, the method comprising:
  • the amplification comprises whole genome amplification.
  • nucleic acids extracted according to the first aspect of the invention have been demonstrated to lead to the generation of amplicons having a smaller size and which are more representative of the original genomic DNA.
  • the amplicons produced according to the second aspect of the invention may be used in a comparative genomic hybridisation method.
  • the use of amplicons produced according to the second aspect of the invention in a method for comparative genomic hybridisation can increase the sensitivity, reproducibility and/or reliability of the results generated in the comparative genomic hybridisation method.
  • the present invention also provides a kit for performing the method of the first aspect and/or second aspect of the invention, the kit comprising a protease together with instructions for performing the method of the first aspect and/or second aspect of the invention.
  • the present invention provides a method for extracting a nucleic acid from a sample comprising one or more cells containing the nucleic acid, the method comprising the steps of:
  • the present invention contemplates a method for extracting a nucleic acid from a sample comprising one or more cells containing the nucleic acid.
  • the method of the present invention is adapted to the extraction of a nucleic acid from a single cell.
  • the sample comprises a single cell.
  • the cell or cells for use in the method may be any suitable eukaryotic or prokaryotic cell.
  • suitable eukaryotic cells include animal, plant or fungal cells, while examples of suitable prokaryotic cells include bacterial or archaeal cells.
  • the one or more cells comprise an animal cell. In some embodiments, the one or more cells comprise a mammalian cell. In some embodiments the one or more cells comprise a human cell.
  • the one or more cells may be derived from a multicellular organism.
  • the one or more cells may be somatic cells derived from an adult form of the organism. Methods for the isolation of various types of somatic cells from an organism are well known in the art and the present invention contemplates any such methods.
  • the one or more cells may be derived from an immature form of the organism and/or may be a germ cell.
  • the cell may be a foetal cell, a cell derived from an embryo (including a blastomere) or a germ cell (including oocytes and sperm).
  • foetal cells examples include a foetal cell taken from the amniotic fluid surrounding the foetus, a foetal cell taken from the maternal circulation, or a foetal cell taken from the mother's reproductive tract (eg. cervical or vaginal lavage).
  • Foetal blood cells unlike mature blood cells, are nucleated and may be isolated from the maternal circulation on the basis of this nucleation.
  • a small number of cells may be removed from an embryo.
  • one or more cells in an embryo may be removed by cleavage stage embryo biopsy. This procedure is usually performed on day 3 of development, when the embryo is at the 6-8 cell stage.
  • the biopsy consists of two stages. The first is to make a hole in the zona pellucida that surrounds the embryo at this time, usually using acid Tyrodes solution or a non-contact laser. Once the hole is made, the cell may then be removed from the embryo.
  • the germ cell for example an oocyte or sperm cell
  • the germ cell may be analysed directly.
  • a polar body from the oocyte may be isolated.
  • Examples of suitable methods for the isolation of embryonic cells, foetal cells or germ cells include:
  • amniocentesis and/or chorionic villous sampling for example as described by Nagel et al. (Prenatal Diagnosis 18: 465-475, 1998); and
  • the method of the present invention contemplates lysing the one or more cells in the sample to release the nucleic acid from the one or more cells into a supernatant.
  • the present invention contemplates any suitable method for the lysis step. Examples of suitable cell lysis or cell disruption methods include:
  • osmosis based methods wherein lowering the ionic strength of culture media causes cells to swell and burst;
  • enzymatic methods including digestion using enzymes such as lysozyme, lysostaphin, zymolase, cellulase, mutanolysin, glycanases, proteases, mannase and the like;
  • mechanical cell disruption methods including, for example, beadbeating, rotor stator processors, valve type processors, French presses and the like;
  • detergent based cell lysis including the use of nonionic or zwitterionic detergents such as CHAPS, and the Triton X series of detergents, or the use of ionic detergents such as SDS;
  • the cells are lysed in the absence of added protease.
  • lysing the one or more cells in the sample comprises alkaline lysis of the one or more cells in the sample.
  • alkaline lysis refers to lysis of a cell in a strongly alkaline solution.
  • Reference herein to a "strongly alkaline” solution includes, for example, a solution having a pH of greater than 7, 8, 9, 10, 11, 12 or 13.
  • strongly alkaline solutions may include solutions of bases such as sodium hydroxide, potassium hydroxide and the like.
  • alkaline lysis may also be performed in the presence of a detergent or surfactant.
  • alkaline lysis may also be performed in the presence of a deprotecting agent for thiolated DNA.
  • a deprotecting agent includes dithiothreitol (DTT).
  • the alkaline lysis comprises lysis in a buffer comprising potassium hydroxide and DTT.
  • the alkaline lysis buffer comprises potassium hydroxide at a concentration of about 100 mM, about 150 mM, about 200 mM, about 250 mM, about 300 mM or about 400mM.
  • the alkaline lysis buffer comprises DTT at a concentration of about about 25 mM, about 50 mM, about 75 mM, or about 100 mM.
  • the method further comprises a neutralisation step prior to contacting the supernatant with the protease.
  • a neutralisation step might include the addition of a neutralisation solution containing a buffer (eg. Tris) to the supernatant.
  • the neutralisation solution may also include an acid (eg. HC1).
  • the present invention contemplates contacting the supernatant containing the nucleic acid with a protease in order to digest one or more proteins associated with the nucleic acid.
  • a protease may be any enzyme that conducts proteolysis.
  • proteases begin protein catabolism by hydrolysis of the peptide bonds that link amino acids together in a polypeptide chain.
  • the protease comprises an endopeptidase.
  • an "endopeptidase” should be understood as any protease which hydrolyses a peptide bond between nonterminal amino acids within a polypeptide or protein.
  • the protease comprises a serine protease (or serine endopeptidase).
  • a "serine protease” or “serine endopeptidase” should be understood to include any protease or endopeptidase in which one of the amino acids at the active site is serine.
  • Serine proteases may be grouped into clans that share homology and may be further subgrouped into families with similar sequences. The major clans found in humans include the chymotrypsin-like, the subtilisin-like, the alpha/beta hydrolase, and signal peptidase clans.
  • the protease comprises a serine protease of the chymotrypsin- like clan.
  • Three exemplary groups of serine proteases of the chymotrypsin-like clan include the chymotrypsins, trypsins, and elastases. All of these enzymes are similar in structure, although they cleave polypeptides at different sites:
  • Chymotrypsins cleave peptide bonds following a bulky hydrophobic amino acid residue.
  • Preferred residues include phenylalanine, tryptophan, and tyrosine, which fit into a hydrophobic pocket in the active site of the enzyme. Trypsins cleave peptide bonds following a positively-charged amino acid residue. Instead of having the hydrophobic pocket of a chymotrypsin, there exists an aspartic acid residue at the base of the pocket. This residue can interact with positively- charged residues such as arginine and lysine on the substrate peptide to be cleaved.
  • Elastases cleave peptide bonds following a small neutral amino acid residue, such as alanine, glycine, and valine.
  • the pocket that is in trypsin and chymotrypsin is now partially filled with valine and threonine, rendering it a mere depression, which can accommodate smaller amino acid residues.
  • the protease comprises a trypsin.
  • Trypsins predominantly cleave peptide chains at the carboxyl side of the amino acids lysine or arginine, except when either is followed by proline. Trypsins have an optimal operating pH of about 8 and optimal operating temperature of about 37°C. A range of exemplary trypsin proteins are described under Enzyme Commission code EC 3.4.21.4 (eg. see http://www.expasy.Org/cgi-bin/nicezyme.pl73.4.21.4).
  • the method of the first aspect of the invention contemplates contacting the supernatant containing the nucleic acid with a protease under conditions suitable for the protease to digest one or more proteins associated with the nucleic acid.
  • suitable conditions may include a pH of about 8, and a temperature of about 37°C. Suitable incubation times will also be ascertained by a person skilled in the art.
  • the method further comprises the step of inhibiting or substantially inactivating the protease at a time after contacting the protease with the supernatant. Inhibition and/or substantial inactivation of the protease may be desirable where the extracted DNA is later used in a further enzymatic process, for example DNA amplification or restriction digestion.
  • any suitable method for the inhibition and/or substantial inactivation of the protease may be used.
  • organic and/or inorganic inhibitors of the enzyme may be added.
  • the conditions in the supernatant may be altered to effect inhibition and/or substantial inactivation of the protease, for example, the temperature, pH, salinity or the like may be adjusted to outside the active range of the protease.
  • inhibiting and/or substantially inactivating the protease comprises the addition of a divalent cation at a concentration sufficient to inhibit and/or substantially inactivate the protease.
  • suitable divalent cations may include Mg 2+ and Ca 2+ .
  • the divalent cation may be added as a salt which includes the divalent cation, for example, Mg 2+ may be added in the form a magnesium salt such as MgCh.
  • inhibiting and/or substantially inactivating the protease comprises holding the supernatant for a time and at a temperature sufficient to inhibit and/or substantially inactivate the protease. Suitable times and temperatures sufficient to inhibit and/or substantially inactivate a particular protease would be readily ascertained by a person skilled in the art. However, by way of example, temperatures of at least 50°C, 55°C, 60°C or 65°C held for at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 minutes may be sufficient to inhibit and/or substantially inactivate proteases such as trypsin.
  • the present invention also provides a method for amplifying a nucleic acid, the method comprising:
  • “Amplifying" a nucleic acid includes any in vitro use of an enzyme to replicate a specific target nucleic acid to a level at which it may be detected.
  • Examples of amplification include polymerase chain reaction (PCR), transcription mediated amplification (TMA), nucleic acid sequence based amplification (NASBA), random amplification (as discussed below), various whole genome amplification methods (as discussed below) and the like.
  • the amplification comprises random amplification.
  • Random amplification may include randomly primed amplification using one or more primers including a sequence of one or more random nucleotides, the sequence of random nucleotides being sufficiently long so as to enable the primer to hybridize to the target nucleic acid under selected conditions at random positions and serve as a primer for extension by a polymerase.
  • the primer may be a primer including a stretch of six or more contiguous nucleotides of random sequence.
  • random amplification may also be achieved with one or more primers of defined sequence, but wherein at least some cycles of the amplification reaction have sufficiently low stringency to enable random binding of the one or more primers to the template.
  • a low number of cycles of amplifcation may be performed under low stringency conditions that allow the one or more primers to prime synthesis randomly throughout the target, followed by a second stage amplification performed under more stringent conditions for generally a larger number of cycles.
  • the amplification comprises whole genome amplification.
  • Whole genome amplification as referred to herein should be understood to include the amplification of a genomic DNA template in an aspecific way, in order to generate amplicons that are representative of the original genomic DNA but with a higher copy number of one or more representative regions of the genomic DNA.
  • PEP Primer Extension PCR
  • DOP-PCR Degenerated Oligonucleotide Primed PCR
  • LMP Ligation Mediated PCR
  • T7-based linear amplification of DNA which is a variant of a protocol originally designed to amplify mRNA that has been adapted for WGA. It uses Alu I restriction endonuclease digestion and a terminal transferase to add a polyT tail on the 3' terminus. A primer is then used with a 5' T7 promoter and a 3' polyA tract, and Taq polymerase is used to synthesise the second strand. Then the sample is submitted to in vitro transcription and posterior reverse transcription; and Multiple displacement amplification (MDA) is a non-PCR-based isothermal method based on the annealing of random hexamers to denatured DNA, followed by strand-displacement synthesis at constant temperature.
  • MDA Multiple displacement amplification
  • the reaction can be catalysed by the Pfe ' 29 DNA polymerase or by the large fragment of the Bst DNA polymerase.
  • the amplification, or whole genome amplification comprises DOP-PCR.
  • the DOP-PCR comprises the use of an oligonucleotide primer comprising includes six or more contiguous nucleotides of random sequence.
  • the one or more primers comprise a primer with the following nucleotide sequence: C C GAC T C G AGNNNNN AT GT GG (SEQ ID NO: 1); wherein each N is independent and each may be any nucleotide.
  • protease treatment of the nucleic acid digests one or more proteins associated with the nucleic acid and thus improves access for a nucleic acid primer and/or polymerase to the template nucleic acid.
  • additional primer binding sites are made available to amplification primers and/or polymerase.
  • the increased availability of primer binding sites in a genomic DNA template has the effect of making the primer binding sites generally closer together. This, in turn, has the effect of both decreasing the average size of the amplicons and making the amplicons generally more representative of the original genomic DNA template.
  • the second aspect of the invention should not be considered limited to random amplification and/or whole genome amplification. Rather, the second aspect of the invention contemplates any nucleic acid amplification wherein contacting the template nucleic acid with a protease under conditions suitable for the protease to digest one or more proteins associated with the nucleic acid would be desirable. As such, amplification may also include any nucleic acid amplification reaction, wherein the protease treatment of the template nucleic acid improves access of a polymerase and/or primer to the template nucleic acid. In some embodiments, the method of the second aspect of the invention may be useful where a nucleic acid template is recalcitrant to amplification due to the presence of protein associated with the nucleic acid.
  • the amplicons produced according to the second aspect of the invention may be used for any purpose.
  • the amplicons produced according to the second aspect of the invention may be used in a comparative genomic hybridisation method.
  • the present invention provides a comparative genomic hybridisation method comprising the use of an amplicon produced according to the second aspect of the invention.
  • the amplicons may be used as immobilised nucleic acids on a solid substrate and/or the amplicons may be labelled and hybridised to an immobilised nucleic acid.
  • Methods for performing comparative genomic hybridisation are well known in the art and the present invention contemplates the use of the amplicons in any suitable method.
  • an exemplary method for comparative genomic hybridisation is described in WO 2004/088310, the content of which is hereby incorporated by reference.
  • the use of amplicons produced according to the second aspect of the invention in a method for comparative genomic hybridisation can increase the sensitivity, reproducibility and/or reliability of the results generated in a comparative genomic hybridisation method.
  • a trypsin digestion step was used in the cell lysis step of a comparative genomic hybridisation method
  • the rate of false negative, false positive and Y diagnosis improved in the comparative genomic hybridisation.
  • a decrease in average slide standard deviation (S.D.) which is an indicator of the quality of amplification and hybridisation, was also observed in a comparative genomic hybridisation which used trypsin digestion in the cell lysis step.
  • the present invention also provides a kit for performing the method of the first aspect and/or second aspect of the invention, the kit comprising a protease together with instructions for performing the method of the first aspect and/or second aspect of the invention.
  • the protease included in the kit may be a protease as described with reference to the first aspect of the invention.
  • the protease may comprise any of an endopeptidase, a serine protease, a serine protease of the chymotrypsin-like clan or trypsin.
  • the kit further comprises a buffer suitable for alkaline lysis of a cell, as hereinbefore described.
  • the buffer comprises potassium hydroxide and DTT.
  • the kit further comprises one or more reagents for the amplification of a nucleic acid.
  • the one or more reagents comprise a DNA polymerase.
  • a range of DNA polymerases would be readily ascertained by a person of ordinary skill in the art and the kit of the present invention may include any suitable DNA polymerase.
  • the DNA polymerase included in the kit may be a DNA polymerase suitable for nucleic acid amplification, including random amplification and/or whole genome amplification as hereinbefore described.
  • the one or more reagents comprise a nucleic acid primer.
  • the primer(s) may be suitable for nucleic acid amplification, including random amplification and/or whole genome amplification as hereinbefore described.
  • the kit may also include additional reagents for the amplification of a nucleic acid including, for example, buffers, dNTPs, one or more restriction endonucleases (eg. as used in some WGA methods), and the like.
  • additional reagents for the amplification of a nucleic acid including, for example, buffers, dNTPs, one or more restriction endonucleases (eg. as used in some WGA methods), and the like.
  • the kit may also comprise one or more reaction vessels in which the methods of the present invention may be performed.
  • reaction vessels in which the methods of the present invention may be performed.
  • Figure 1 shows DOP-PCR products amplified from DNA extracted from single human cells using the standard lysis method (lanes 2-17 of the top row), the extended lysis method (lanes 18 to 28 top row and lanes 3 to 6 bottom row) and an embodiment of the lysis method of the present invention utilizing a trypsin digestion step (lanes 7 to 22 of the bottom row).
  • Lane 1 in each of the top and bottom rows shows an Sppl marker, while lane 29 in the top row and lane 24 in the bottom row shows a pUC19 marker.
  • Figure 2 shows the results of CGH using DNA extracted from single human cells using the control cell lysis method.
  • Figure 3 shows the results of CGH using DNA extracted from single human cells using the extended lysis cell lysis method.
  • Figure 4 shows the results of CGH using DNA extracted from single human cells using a cell lysis method according to an embodiment of the present invention, which incorporates a trypsin digestion step.
  • Alkaline Lysis Buffer 200mM KOH, 50mM DTT was added to a single cell in a PCR tube. 5 ⁇ of Alkaline Lysis Buffer was used for a final first round DOP-PCR reaction volume of 50 ⁇ .
  • the mixture was then incubated at 65°C for 5 min followed by 6°C for 5 min.
  • the mixture was incubated at 65°C for 10 min.
  • Neutralization Buffer 3 500mM Tris-HCl pH 8.3, 200mM HC1 was added. 5 ⁇ of Neutralization Buffer 3 was added for a final first round DOP-PCR reaction volume of 50 ⁇ .
  • ⁇ of 8mg/mL stock trypsin (Porcine trypsin 1:250 MP Biomedicals Cat # 103139) was added to the cell lysate after the addition of neutralisation buffer 3. This mixture was then incubated at 37°C for 10 minutes. After incubation, 3 ⁇ of 25mM MgCh is added to the lysate. The trypsin was then further inactivated by incubation at 65°C for 10 min followed by 6°C for 4 minutes.
  • PCR reaction mixes were prepared as follows (/50 ⁇ 1): Ultra-Pure H2O - 20.6 ⁇ 1, 0.1% Gelatin - 5.0 ⁇ 1, dNTPs (2.5mM each) - 5.0 ⁇ 1, DOP PCR primer (20 ⁇ ) - 5.0 ⁇ 1, KAPA 2G Robust (5 ⁇ / ⁇ 1) - 0.4 ⁇ 1, lysed single cell (template) - 14 ⁇ 1.
  • a PCR mastermix for labelling with DY547 was prepared as follows (/24 ⁇ 1): 11.55 ⁇ 1 of Ultra-Pure H2O; 5.0 ⁇ 1 of 5X KAPA 2G buffer A; 2.5 ⁇ 1 of 0.1% Gelatin; 2.5 ⁇ 1 of DOP- PCR primer (20 ⁇ ); 1.5 ⁇ 1 of DY547 dNTP labelling mix (3:1); 0.2 ⁇ 1 of K2GR (5 ⁇ / ⁇ 1); and 0.75 ⁇ of DY547-dUTP.
  • a PCR mastermix for labelling with DY647 was prepared as follows (/24 ⁇ 1): 11.70 ⁇ 1 of Ultra-Pure H2O; 5.0 ⁇ 1 of 5X KAPA 2G buffer A; 2.5 ⁇ 1 of 0.1% Gelatin; 2.5 ⁇ 1 of DOP- PCR primer (20 ⁇ ); 1.5 ⁇ 1 of DY647 dNTP labelling mix (4:1); 0.2 ⁇ 1 of K2GR (5 ⁇ / ⁇ 1); and 0.6 ⁇ of DY647-dUTP.
  • Labelled DNA mixture for hybridization was prepared in a sterilized 0.5ml PCR tube (MBP tube) by combining the following: 35 ⁇ of human Cot-1 DNA (lmg/ml - final amount 35 ⁇ g), 20 ⁇ of sheared salmon sperm DNA (lmg/ml - final amount 20 ⁇ g), ⁇ , of DY547-labelled DNA ( ⁇ l ⁇ g), ⁇ , of DY647-labelled DNA ( ⁇ l ⁇ g), 7.5 ⁇ of NaAC, pH 5.2 (3M), 150 ⁇ of 100% ethanol.
  • MBP tube sterilized 0.5ml PCR tube
  • the tube containing the DNA mixture was incubated at -20°C for 1 hour in the dark. DNA pellets were then collected by centrifugation at 10,000 g for 25 min at 4°C. The ethanol was then decanted. The DNA pellets were then washed with 200 ⁇ 1 of 75% ethanol, followed by centrifugation at 10,000 g for 5 min at 4°C and removal of the ethanol supernatant. DNA pellets were then dried at 50°C for ⁇ 5 min in a heating block. The dried DNA pellets were then redissolved by adding ⁇ of hybridisation solution to each dried DNA pellet. This mixture was then incubated at 37°C for 5 min. After incubation the tubes were mixed and briefly centrifuged. The incubation, mix and brief centrifugation were repeated. The DNA probes were then denatured at 80°C for 10 min in a PCR machine followed by pre-annealing the probes at 37°C for 30 min (Corbett PC9606C; program 80). It) Blocking Slides
  • Array slides were immersed in blocking buffer (Blocklt; Arraylt Corporation, Sunnyvale, California, Cat # BKT) and left at room temperature for a minimum of lh and no longer than 24h (typically 2h), on an orbital mixer. The slides were then removed from the blocking buffer, and rinsed for 1 min under running RO H2O. The slides were then centrifuged (array side up) until dry in an Arraylt centrifuge. The dried slides were then stored at room temperature, in a slide box wrapped with aluminium foil until required. Blocked slides remained useable for several hours.
  • blocking buffer Blocklt; Arraylt Corporation, Sunnyvale, California, Cat # BKT
  • a hybridisation chamber (Corning) was disassembled and the base, the cover, and metal clips were placed on a warming tray. An array slide (with the printed array slide up) and coverslips were also placed on the warming tray. The disassembled hybridisation chamber and the array slide were then incubated for 30-60 min on the warming tray.
  • the array slide was overlaid on an array template. 10.5 ⁇ 1 of pre- annealed probes were pipetted onto the array area of the array slide sitting on the warm tray and then covered with a coverslip. The array slide (DNA up) was then placed in the base of the hybridisation chamber. 10 ⁇ of PCR water was then pipetted into the humidifying well at each end of the chamber base. The cover of the chamber was then placed over the base and secured with clips. The hybridisation chamber was then incubated at 37°C for 16-20 hrs. iv) Post-Hybridization Washing
  • Hybridised slides were removed from the hybridisation chamber and then sequentially immersed in the following: 50% formamide/2X SSC at room temperature in the dark until the coverslip slid off;
  • slides were then dried using a microarray high-speed centrifuge (TeleChem International, Inc.). After drying, slides were either scanned immediately or stored in a slide box, wrapped in foil at room temperature in the dark for up to 2 months.
  • TeleChem International, Inc. a microarray high-speed centrifuge
  • Single human cells were selected for controls, extended lysis, or trypsin digestion.
  • the cells for the extended lysis test were processed first: cells were lysed as described above in RHS protocol 10, but with the lysis step extended to 10 minutes, before being neutralised and then stored at 4°C until PCR mastermix was added.
  • Control cells and cells for trypsin digestion were processed next: the cells were lysed for 5 minutes and then neutralised as described above in RHS protocol 010. The control cells were then stored at 4°C until mastermix was added. As described above, the cells for trypsin digestion were then treated with trypsin, incubated for 10 minutes at 37°C. Magnesium chloride was added to block/slow the action of trypsin and then these samples were incubated at 65°C for 10 minutes to further inactivate the trypsin, the samples were then cooled. The appropriate mastermix was then added to the cells and the samples were placed in the FlexCycler (0.5mL block - Analytik Jena AG) and amplified as described above under RHS protocol 011.
  • DNA was extracted from single human cells of known karyotype using each of the control, extended lysis and trypsin digestion methods described above. The extracted DNA was then amplified and used for comparative genomic hybridisation (CGH). The CGH method was performed according to RHS protocol 014 as described above.
  • the rate of false positive/negatives was decreased when compared to the control cells.
  • the improvement in FP/FN rates was not as great as that seen with the trypsin digestion treatment.

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Abstract

La présente invention porte d'une façon générale sur des procédés pour l'extraction d'un acide nucléique à partir d'un échantillon comprenant une ou plusieurs cellules contenant l'acide nucléique. La présente invention porte également sur l'amplification des acides nucléiques extraits, en particulier sur l'amplification des acides nucléiques extraits par la technique d'amplification du génome entier. Les procédés de la présente invention sont généralement fondés sur la lyse d'une ou plusieurs cellules dans un échantillon pour libérer un acide nucléique et la mise en contact de l'acide nucléique avec une protéase dans des conditions appropriées pour que la protéase digère une ou plusieurs protéines associées à l'acide nucléique.
PCT/AU2010/001151 2009-09-07 2010-09-07 Extraction d'acide nucléique WO2011026194A1 (fr)

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AU2009904275A AU2009904275A0 (en) 2009-09-07 Nucleic acid extraction
AU2009904275 2009-09-07

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CN102321618A (zh) * 2011-09-30 2012-01-18 生工生物工程(上海)有限公司 一种用于从生物材料中直接扩增dna片段的方法及试剂盒
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US11104888B2 (en) 2018-10-31 2021-08-31 Illumina, Inc. Polymerases, compositions, and methods of use
US11001816B2 (en) 2018-12-05 2021-05-11 Illumina, Inc. Polymerases, compositions, and methods of use
US11634697B2 (en) 2018-12-05 2023-04-25 Illumina, Inc. Polymerases, compositions, and methods of use
US12104182B2 (en) 2018-12-05 2024-10-01 Illumina, Inc. Polymerases, compositions, and methods of use
US12077789B2 (en) 2021-08-14 2024-09-03 Illumina, Inc. Polymerases, compositions, and methods of use

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