EP1451361A2 - Verfahren zur detektion und quantitativen analyse von nuklleinsäure molekülen welches einen extraktions-schritt mit einen starken base enthält und anwendungen - Google Patents

Verfahren zur detektion und quantitativen analyse von nuklleinsäure molekülen welches einen extraktions-schritt mit einen starken base enthält und anwendungen

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Publication number
EP1451361A2
EP1451361A2 EP02793232A EP02793232A EP1451361A2 EP 1451361 A2 EP1451361 A2 EP 1451361A2 EP 02793232 A EP02793232 A EP 02793232A EP 02793232 A EP02793232 A EP 02793232A EP 1451361 A2 EP1451361 A2 EP 1451361A2
Authority
EP
European Patent Office
Prior art keywords
dna
biological sample
strong base
cell
nucleic acid
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.)
Withdrawn
Application number
EP02793232A
Other languages
English (en)
French (fr)
Inventor
Gabrielle Potocki
Laure Sabatier
Henri Benech
Jacques Grassi
Jean-Robert Deverre
Marie-Anne Marriere
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.)
Commissariat a lEnergie Atomique et aux Energies Alternatives CEA
Original Assignee
Commissariat a lEnergie Atomique CEA
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 Commissariat a lEnergie Atomique CEA filed Critical Commissariat a lEnergie Atomique CEA
Publication of EP1451361A2 publication Critical patent/EP1451361A2/de
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6851—Quantitative amplification
    • 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
    • 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 to a method for the detection and quantitative analysis of nucleic acid molecules comprising a step extraction and random fractionation of said nucleic acid molecules by a strong base, and its applications.
  • nucleic acid molecules from cell suspensions. These methods generally include a step of cell lysis by enzymatic, osmotic or even chemical treatment, followed by a step of extraction of the nucleic acid molecules generally in alkaline condition.
  • extraction processes there are mainly two main classes of extraction processes, namely the extraction processes making it possible to preserve the integrity of the nucleic acid molecules (DNA) of the cells that the we want to analyze and extraction methods leading to DNA fractionation.
  • DNA extraction methods are often lengthy, and include multiple limiting steps, in particular steps of isolating DNA from other biomolecules such as RNAs or proteins, which are likely to be released during the different processing steps. These isolation steps are necessary so as not to disturb or even prevent subsequent measurements.
  • the presence of unfractionated DNA molecules is not suitable.
  • the viscosity and the size of long nucleic acid molecules are not appropriate or else require the use of denaturation or hybridization conditions which are not suitable for a given type of detection.
  • the DNA extraction methods must make it possible to fractionate the DNA molecules.
  • the first object of the invention is therefore a method for the detection and quantitative analysis of nucleic acid molecules from a biological sample, characterized in that it comprises at least one phase of extraction of said acid molecules nucleic acids, said extraction phase comprising the following steps: a) bringing a biological sample comprising said nucleic acid molecules into contact with a strong base having sufficient molarity to give the biological sample a pH greater than 13 , b) lysis of the mixture obtained until complete dissolution of the biological sample, and c) neutralization of the reaction medium, to obtain fragments of neutralized random nucleic acid molecules.
  • This process has many advantages. It is simple, inexpensive, easy to implement and perfectly reproducible. It does not carry out dangerous manipulations such as the use of X-rays. It can also be used on a large scale. Furthermore, the random fragments obtained by implementing this method have the advantage of being able to be directly used in methods of quantitative analysis of DNA, that is to say without compulsory additional purification step.
  • the biological sample consists for example of suspensions of cell pellets, of DNA purified from cell cultures or of tissue homogenates.
  • the cell concentration within said suspension is preferably between 5.10 and 1.10 cells / ml of strong base and even more particularly between 5.10 '"' and 3.10 ' ' cells / ml of strong base.
  • the concentration of DNA is preferably between 3.10 " and 0.6 mg of DNA / ml of strong base and even more particularly between 3.10 " 3 and 0.3 mg of DNA / ml of strong base.
  • the nature of the strong base used in accordance with the invention is not critical. It can in particular be chosen from soda and potash; soda being particularly preferred.
  • the molarity of the strong base enabling the pH of the biological sample to be brought to a pH value greater than 13 is generally between 0.7 and 1.5 M, a molarity of 1 M being very particularly preferred.
  • Stage b) of lysis is preferably carried out for a period of between 15 minutes and 24 hours.
  • This contact time must of course be adapted to the nature of the biological sample to be analyzed.
  • the duration of the lysis step is preferably between 3 and 12 hours, so as to allow complete dissolution of the sample .
  • the duration of lysis can be shorter and is generally between 15 minutes and 2 hours.
  • the lysis step is preferably carried out at a temperature between room temperature and 100 ° C, and preferably between room temperature and 40 ° C.
  • the extraction phase of the process in accordance with the invention may also comprise an additional step of sonication of the sample, so as to accelerate the lysis step.
  • This additional sonication step is preferably carried out after step a), within approximately 15 to 30 minutes following the contacting of the sample with the strong base.
  • the sonication stage can be carried out one or more times, generally once or twice, for durations varying between 15 and 30 minutes.
  • the cell lysis step proper is then continued normally until the sample is completely dissolved.
  • This sonication step is not necessary when the sample consists of purified DNA.
  • the pH of the reaction medium is then preferably brought to a value of between 6 and 8 approximately and very preferably to a value of approximately 7.5.
  • This neutralization step is preferably carried out by adding an acidifying agent or a buffer making it possible to lower the pH of the reaction medium to the desired value.
  • this neutralization step is preferably carried out by adding 5 to 7 volumes of a buffer, and even more particularly by adding 6 volumes of a buffer, for a basic volume strong.
  • a buffer Preferably 6 volumes of 1 M sodium phosphate buffer are used
  • Neutralization is of course carried out after step b) of lysis and before the step of quantitative analysis of the DNA of the biological samples.
  • the detection and analysis method according to the invention can be used to determine, in absolute value, the quantities of DNA or the lengths tclomeric DNA by techniques known to those skilled in the art such as hybrid hybrid (Chevrier et al, Mol. Cell Probes, 1993, 7, 187-197).
  • the method according to the present invention can also be used for all applications requiring a cell count, for example, the screening of molecules intended to control the telomeric length, which allows diagnostic aid in oncology, d '' ensuring the monitoring of premature aging, monitoring of immunosenescence, choice of treatments and therapeutic monitoring in chemotherapy, development of research in the biology of aging; counting blood or other cells during clinical examinations; monitoring the extension of stem cells in cell therapy and measuring the concentration of a drug such as antiretrovirals in the context of HIV, etc.
  • the size of the random and neutralized fragments of nucleic acid molecules obtained by implementing the method according to the invention is generally between 50 and 50,000 base pairs (bp).
  • the invention therefore also relates to the use of neutralized random fragments in accordance with the invention, in a method of detection and quantitative analysis of DNA, as well as for cell counting.
  • the invention also comprises other provisions which will emerge from the description which follows, which refers to an example of extraction and quantification of DNA from cell extracts and determination of size telomeres of chromosomes of eukaryotic cells, to an example of correlation between the quantity of DNA and the counting of peripheral blood mononuclear cells during the assay of intracellular metabolites, as well as to the appended figures in which:
  • FIG. 1 shows the relationship between fluorescence as a function of the quantity of DNA, in the case of standard DNA (DNA or DNA extracted from CIM diploid cells (cultures of human fibroblasts);
  • FIG. 2 shows the relationship between fluorescence and the amount of cells extracted in 100 ⁇ l of 1 M NaOH, in the case of lysates diluted 10 times;
  • FIG. 3 shows the relationship between fluorescence and the amount of cells extracted from 100 ⁇ l of 1 M NaOH, in the case of lysates diluted 100 times;
  • FIG. 4 shows the absorbance values at 414 nm as a function of the number of telomeric units / ml in the case of a cell lysate of CIM cells;
  • FIG. 5 shows the correlation between the average telomeric lengths determined by implementing the method according to P Invention (ordinate axis) and a determination made by the conventional method of Southern blot (abscissa axis).
  • FIG. 6 shows the relationship between the quantification of the number of cells on Malassez cell (abscissa axis) and the fluorescence signal obtained by the DNA quantification method according to the invention (ordinate axis).
  • EXAMPLE 1 EXTRACTION AND QUANTIFICATION OF THE DNA FROM CELL EXTRACTS AND DETERMINATION OF THE SIZE OF TELOMERES OF EUKARYOTIC CELL CHROMOSOMES I - Material and method 1) Preparation of cell lysates
  • Eukaryotic cells from vertebrates are washed in phosphate buffered saline (PBS), then the pellets are dried, frozen in liquid nitrogen and stored at - 80 ° C or - 20 ° C for subsequent use. After thawing at 4 ° C, the cells are taken up at the rate of 1. l ⁇ / 100 ⁇ l of 1 M NaOH and vortexed. After 2 x 30 minutes of sonication at room temperature, the cell lysates are incubated at room temperature overnight, until the pellet is completely dissolved. The cell lysates are neutralized to pH 7.5 by the addition of 6 volumes of 1 M sodium phosphate buffer (pH 7) for 1 volume of 1M NaOH.
  • PBS phosphate buffered saline
  • the lysates are previously partially desalted by diafiltration. After centrifuging the samples (200 ⁇ l), at 13,000 g, for 15 minutes and at 20 ° C, in Microcon®-30 (Amicon), the retentates are diluted in 200 ⁇ l of water, then centrifuged again at 10 000 g, for 5 minutes at 20 ° C. The retentates are recovered and deposited on a new Microcon®-30, then centrifuged at 10,000 g, for 5 minutes at 20 ° C.
  • the samples (105 ⁇ l of final volume) are diafiltered, partially desalted and concentrated 6.7 times.
  • the size of the DNA fragments can then be determined by electrophoresis on 2% agarose gel.
  • the cellular DNA fragmented during the sodium hydroxide extraction is quantified by fluorescence using a Fluoroscan®II fluorimeter (Labsystems).
  • the sample consists of 10 ⁇ l of neutralized cell lysate or 10 ⁇ l of standard solution (DNA ladder XIV, Roche, at 250 ng / ⁇ l or DNA ladder 200 bp at 250 ng / ⁇ l, Roche) and 100 ⁇ l of reagent SYBr Green (Interchim, Molecular Probes, SYBR Gold nucleic acid, Ref. SI 1494) diluted extemporaneously to 1 / 10,000 in water.
  • the linearity range of the method is between 0.1 and 10 ng of DNA.
  • the reading is carried out after one hour of incubation at room temperature (between 20 and 25 ° C), by excitation of the intercalating agent at 465 nm and the emission reading is carried out at 530 nm.
  • the method consists, firstly, in covalently fixing on a plate, for example a 96-well plate, an oligonucleotide sensor complementary to the telomeric sequences.
  • the telomeric sequence consists of a repeat of the TTAGGG motif.
  • the sensor oligonucleotide (SflA8) therefore has the sequence A8 (TTAGGG) 6 , on which the telomeric sequences contained in the neutralized cell lysates are fixed by complementarity.
  • a tracer oligonucleotide (SflCSh) having the sequence (TTAGGG) 3 coupled to the biotin at 3 ′ is then hybridized to the whole.
  • the detection is carried out by colorimetry, via a coupling between streptavidin (which has an affinity for biotin) and acetylcholmesterase, which pemiet the transfomiation of a colorless substrate, the Ellman reagent, into a colored product.
  • streptavidin which has an affinity for biotin
  • acetylcholmesterase which pemiet the transfomiation of a colorless substrate, the Ellman reagent
  • the oligonucleotide SflA8 at 100 nmol / ml (1,270 ⁇ g / ml) is incubated for 10 minutes at a temperature of 95 ° C., then for 10 minutes in ice.
  • the sensor oligonucleotide (160 ⁇ l) is then diluted in a solution consisting of: - 6 ml of a 100 mM solution of 1-methyl-imidazole (1-MeLM), pH 7, (prepared by diluting 398.6 ⁇ l 1-MeIM in 220 ⁇ l of fuming HCl and a sufficient quantity of water to have a final volume of 50 ml, said solution having been filtered on a 0.45 ⁇ m filter),
  • the sensor oligonucleotide is then incubated overnight at 50 ° C. in a 96-well plate comprising surface -NH functions (Covalent Binding NUNC®, Covalink), at a rate of 100 ⁇ l per well.
  • the covalent attachment of the oligonucleotide to the plate is therefore carried out by means of the terminal phosphate groups in position 5 ′ of the sensor oligonucleotide, via the amino groups using PEDAC.
  • the plate is then washed 3 times with the washing buffer (0.4 M NaOH; sodium dodecylsulfate (SDS) 10 mM), preheated to 50 ° C., at a rate of 200 ⁇ l per well, incubated for 5 minutes at 50 ° C., then washed 2 more times.
  • the washing buffer 0.4 M NaOH; sodium dodecylsulfate (SDS) 10 mM
  • the plate is then washed with deionized water and then stored at 4 ° C. ? . ) . - . 9 s age of .the .length of . tel mothers
  • oligonucleotide solutions, standards lysates and cell lysates were incubated for 10 minutes at 60 ° C and then for 15 minutes at 4 ° C sui ⁇ ice.
  • 50 ⁇ l of tracer oligonucleotides SflC ⁇ h at 10 nmol / ml are diluted in 4,950 ⁇ l of hybridization buffer (TH: 5 mM sodium phosphate buffer, pH 7.0, 0.75 M NaCl, 5 mM EDTA, solution filtered through 0.45 ⁇ m filter).
  • telomeric length a relationship is established between the absorbance at 414 nm (A 4
  • the 46 chromosomes of a diploid cell represent a mass of 6 ⁇ 10 -12 g.
  • Chromosomes / ml C x 46 / 6.10 ""
  • L (bp) (telo units / ml) x 6 / [(chromosomes / ml) x2]
  • telomeric lengths were also measured by the conventional hybridization method of the Southern blot type.
  • the DNA is purified from 10 6 cells by phenol / chloroform extractions according to the methods commonly used by those skilled in the art.
  • To estimate the size of the telomeric repeats 2 ⁇ g of DNA sample are digested with Rsal and Hinfl restriction endonucleases (Biolabs) at a concentration of 5 units / ⁇ g of DNA for 2 hours at a temperature of 37 ° C. .
  • the DNA sample thus digested is then separated by electrophoresis in a 0.6% agarose gel for 18 hours, then transferred to a nylon membrane, and finally fixed to the membrane under ultraviolet radiation.
  • the membrane is then hybridized with a telomeric probe
  • Hybridization is carried out at 50 ° C for at least 16 hours.
  • the membrane is then washed twice with 2x SSPE buffer (0.2 M Phosphate Buffer, 2.98 M NaCl, 0.02 M EDTA, pH about 7.4) -SDS 0.1% and exposed overnight.
  • phosphorus detection cassette (Storm).
  • the images are acquired using NIH image V 1.6.2 software. and the position of the size markers (1 kb Ladder and ⁇ -Hindffl) is plotted on the image thus obtained.
  • the migration distance is expressed in pixels and the signal intensity in gray levels.
  • the densitometric profile of the telomeric fragments is then quantified using the software.
  • Figures 2 and 3 in the appendix which represent the quantity of DNA extracted dosed by fluorescence (on the ordinate) as a function of the number of cells in 100 ⁇ l of 1 M NaOH (on the abscissa), in the case of neutralized lysates diluted 10 times
  • telomere lengths obtained by implementing each of the two techniques described above have been reported in FIG. 5 appended.
  • the average length of the telomeres (in base pairs) determined according to the process in accordance with the invention is compared to that obtained by the classic Southem blot method (abscissa axis).
  • all of the reagents used in this example can be in the form of a device with several compartments (kit) comprising at least one standard DNA solution, an oligonucleotide sensor, a tracer oligonucleotide, a dilution buffer such as potassium phosphate buffer, a strong base such as 1 M sodium hydroxide, a fluorescent substrate such as SYBr Green, a hybridization buffer such as by example that described above, a streptavidin: enzyme complex such as G4-Slr described above, Ellman reagent and one or more microtiter plates.
  • kit comprising at least one standard DNA solution, an oligonucleotide sensor, a tracer oligonucleotide, a dilution buffer such as potassium phosphate buffer, a strong base such as 1 M sodium hydroxide, a fluorescent substrate such as SYBr Green, a hybridization buffer such as by example that described above, a streptavidin: enzyme complex such as G4-Slr
  • the oligonucleotide can be grafted onto the microtiter plates.
  • the kit as described above can be used during the process according to the invention, to determine, in absolute value, the quantities of DNA or the telomeric lengths of the DNA in a biological sample.
  • EXAMPLE 2 CORRELATION BETWEEN THE QUANTITY OF DNA AND THE COUNTING OF PBMCs DURING THE DETERMINATION OF INTRACELLULAR METABOLITES OF INTIs.
  • PBMCs peripheral blood mononuclear cells
  • PBMCs are isolated from three different blood bags supplied by the French Blood Establishment (samples 1 to 3) using CPT tubes (Becton-Dickinson, ref. 332761) and according to the technique indicated by the manufacturer .
  • the number of PBMCs isolated is measured by counting with a Malassez cell. This counting is carried out from two dilutions of the suspension of PBMCs (l / 20 th and l / 40 th ), by depositing 20 ⁇ l of each previous dilution and counting in duplicate on 10 tiles.
  • QC # 1 Three QCs will be included in each analysis series, for example a QC # 1, a QC # 3 and a QC # 5.
  • Standard 1 and the QCs are then centrifuged at approximately 1,200 g for 3 minutes at 4 ° C. After removal of the supernatant, the standard and QC pellets are dry frozen at -80 ° C.
  • the preparation of samples from HIV positive patients is carried out from a blood sample (about 8 ml) PBMCs, using tubes
  • the PBMCs isolates are then centrifuged at approximately 1,200 g for 3 minutes at 4 ° C. After removal of the supernatant, the pellets of PBMCs are dry frozen at -80 ° C.
  • the cell lysis is then stopped by adding 500 ⁇ l of 1 M potassium phosphate buffer of pH 7.4.
  • the concentrations of the standards obtained are then as follows: 25.10 °; 12.5.10 6 ; 6.25.10 6 ; 3.13.10 e ; 1.56.10 6 and 0.78.10 6 cells per standard.
  • the cell lysate sample (DNA extract) is diluted 1/10 in potassium phosphate buffer. Then deposited at the bottom of a well of an opaque microtiter plate (MUNCH®, Brand Product), 10 ⁇ l of DNA extract diluted with MUNCH®, Brand Product.
  • the reading is done after 60 minutes using a Fluoroscan® II, Labsystems fluorimeter (excitation 465 nm, emission 530 nm).
  • Malassez is plotted on the abscissa axis (number of cells x 10 6 ) and the fluorece ⁇ ce signals obtained according to the method of quantification of conforming DNA are plotted on the ordinate axis.
  • the calculated values for the samples must be within the range of the range. For each sample, the measurement is made 4 times, each measurement taking place on different days.
  • all of the reagents used in this example can be in the form of a device with several compartments (kit) comprising at least one calibration standard consisting of a known quantity of PBMCs, a quality control consisting of a known quantity of PBMCs, a lysed quality control consisting of a known quantity of lysed PBMCs, a dilution buffer such as potassium phosphate buffer, a strong base such as sodium hydroxide 1 M, a fluorescent substrate such as SYBr Green and one or more microtiter plates.
  • kit comprising at least one calibration standard consisting of a known quantity of PBMCs, a quality control consisting of a known quantity of PBMCs, a lysed quality control consisting of a known quantity of lysed PBMCs, a dilution buffer such as potassium phosphate buffer, a strong base such as sodium hydroxide 1 M, a fluorescent substrate such as SYBr Green and one or more microtiter plates.
  • kit as described above can be used during the process according to the invention, for the cell in a biological sample.

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EP02793232A 2001-12-07 2002-11-14 Verfahren zur detektion und quantitativen analyse von nuklleinsäure molekülen welches einen extraktions-schritt mit einen starken base enthält und anwendungen Withdrawn EP1451361A2 (de)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
FR0115843 2001-12-07
FR0115843A FR2833273B1 (fr) 2001-12-07 2001-12-07 Procede de detection et d'analyse quantitative de molecules d'acides nucleiques comprenant une etape d'extraction par une base forte et applications
PCT/FR2002/003889 WO2003048386A2 (fr) 2001-12-07 2002-11-14 Procede de detection et d'analyse quantitative de molecules d'acides nucleiques comprenant une etape d'extraction par une base forte et applications.

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EP1451361A2 true EP1451361A2 (de) 2004-09-01

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US (1) US20050118593A1 (de)
EP (1) EP1451361A2 (de)
AU (1) AU2002358892A1 (de)
FR (1) FR2833273B1 (de)
WO (1) WO2003048386A2 (de)

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US7157561B2 (en) 2001-07-13 2007-01-02 Roche Diagnostics Operations, Inc. Methods of inhibiting transmission of a costimulatory signal of lymphocytes
US7202092B2 (en) 2001-07-13 2007-04-10 Roche Diagnostics Operations, Inc. Indinavir derivatives useful in immunoassay
US20050277121A1 (en) 2004-06-11 2005-12-15 Ambion, Inc. Crude biological derivatives competent for nucleic acid detection
US9556427B2 (en) 2013-02-27 2017-01-31 Syngenta Participations Ag Methods and compositions for preparation of nucleic acids
AU2014381626A1 (en) * 2014-02-04 2015-09-10 Syngenta Participations Ag Genomic DNA extraction reagent and method
US12344884B2 (en) 2018-06-28 2025-07-01 Gen-Probe Incorporated Sample preparation method and system

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DE19724781C2 (de) * 1997-06-12 2003-03-20 Werner E G Mueller Mikro-Methode zur Schnellbestimmung von DNA-Schäden und deren Reparatur unter Verwendung des Fluoreszenzfarbstoffs Picogreen und ihre Anwendung

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AU2002358892A1 (en) 2003-06-17
AU2002358892A8 (en) 2003-06-17
FR2833273B1 (fr) 2004-08-13
WO2003048386A2 (fr) 2003-06-12
WO2003048386A3 (fr) 2004-02-26
US20050118593A1 (en) 2005-06-02
FR2833273A1 (fr) 2003-06-13

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