EP2097537A1 - Procédé spectrométrique de masse sur des échantillons contenant des acides nucléiques - Google Patents

Procédé spectrométrique de masse sur des échantillons contenant des acides nucléiques

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Publication number
EP2097537A1
EP2097537A1 EP07856466A EP07856466A EP2097537A1 EP 2097537 A1 EP2097537 A1 EP 2097537A1 EP 07856466 A EP07856466 A EP 07856466A EP 07856466 A EP07856466 A EP 07856466A EP 2097537 A1 EP2097537 A1 EP 2097537A1
Authority
EP
European Patent Office
Prior art keywords
sample
strand
double
strands
mass
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Withdrawn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
EP07856466A
Other languages
German (de)
English (en)
Inventor
Hüseyin Aygün
Michael Karas
Ute Bahr
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.)
Goethe Universitaet Frankfurt am Main
BioSpring Gesellschaft fur Biotechnologie mbH
Original Assignee
Goethe Universitaet Frankfurt am Main
BioSpring Gesellschaft fur Biotechnologie mbH
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Filing date
Publication date
Application filed by Goethe Universitaet Frankfurt am Main, BioSpring Gesellschaft fur Biotechnologie mbH filed Critical Goethe Universitaet Frankfurt am Main
Priority to EP07856466A priority Critical patent/EP2097537A1/fr
Publication of EP2097537A1 publication Critical patent/EP2097537A1/fr
Withdrawn legal-status Critical Current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
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T436/00—Chemistry: analytical and immunological testing
    • Y10T436/14—Heterocyclic carbon compound [i.e., O, S, N, Se, Te, as only ring hetero atom]
    • Y10T436/142222—Hetero-O [e.g., ascorbic acid, etc.]
    • Y10T436/143333—Saccharide [e.g., DNA, etc.]
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T436/00—Chemistry: analytical and immunological testing
    • Y10T436/25—Chemistry: analytical and immunological testing including sample preparation
    • Y10T436/25125—Digestion or removing interfering materials

Definitions

  • Nucleic acids are composed of individual building blocks, the nucleotides, constructed macromolecules. Alternating monosaccharides and phosphoric acid residues form the backbone of the nucleic acids, with a nucleobase attached to each monosaccharide.
  • Important representatives of the nucleic acids are the deoxyribonucleic acid (DNA), ribonucleic acid (RNA), but also derivatives and modifications thereof, such as the "locked nucleic acid” (LNA), which is a modified RNA.
  • DNA and RNA-like organic polymers, such as e.g. Peptide nucleic acid (PNA) from the preamble of the nucleic acid comprises.
  • nucleic acids An essential property of the nucleic acids is that not only can they be present as single strands of the polymer of nucleotides or derivatives thereof, but also in the form of duplexes or more complex forms (e.g., as a triple helix).
  • a special feature here is that double-stranded or more complex structures are not formed from any individual strands of the nucleic acids, but the formation proceeds depending on the base sequence. Thus, a certain amount of complementarity in the base sequence of two individual nucleic acid strands is necessary in order to form a double-stranded structure.
  • such structures form spontaneously after the nucleic acid single strands have been brought together under physiological conditions (salt concentration, pH and temperature). Double strands are stabilized by hydrophobic interactions within the stacking (stacking interactions) and by intermolecular hydrogen bonding of the respective complementary strands of the single strands.
  • double-stranded nucleic acids are obtained by either separately synthesizing the complementary single strands and then combining under double-stranded conditions under physiological conditions, or synthesizing the double strand in a so-called "tandem" synthesis of the single strand.
  • a linker introduced in the synthesis is hydrolyzed and the resulting single strands, which have complementary base sequences are then hybridized under physiological conditions.
  • RNAi RNA interference or RNA interference
  • siRNAs short double-stranded RNA sequences
  • Elbashir and Tuschl succeeded for the first time in 2001 in proving that such a process can also be induced by means of short double-stranded RNA molecules (siRNAs) (Elbashir SM, Lendeckel W, and Tuschl T, Genes Dev, 2001, 15 (2), 188 Elbashir SM, Harborth J, Lendckel W, Yalcin A, Weber K, Tuschl T, Nature, 2001, 411, 494-498; Caplen NJ, Parrish S, Imani F, Fire A, and Morgan RA, Proc. Natl Acad., USA, 2001, 98, 9746-9747).
  • siRNAs short double-stranded RNA molecules
  • RNA single strands it is above all the receptors TLR 7 and TLR 8 that are able to trigger such an immune response (Heil F, Hemmi H, Hochrein H, Ampenberger F, Kirschning C, Akira S, Lipford G, Wagner H , Bauer S, Science, 2004, 303, 1526-1529). Therefore, applications in which double-stranded nucleic acids act as an active substance should be well characterized in view of the presence of excess single strands.
  • the same proportions of the two complementary single strands are required for the preparation of a double strand.
  • Nucleic acids eg synthetic oligonucleotides, quantified in the prior art by UV-VIS spectroscopy.
  • the extinction coefficients of a solution containing the nucleic acid are measured and the nucleotide content calculated using the Lambert-Beer law.
  • the extinction coefficient of a nucleic acid is approximately additive composed of the extinction coefficients of the individual bases of a strand.
  • the samples obtained which in addition to the proportion of double strand can have a further proportion of single strand, must be characterized in particular by two measured values.
  • the single strand excess (single strand A or complementary single strand B), on the other hand the ratio of single strand to double strand in the sample.
  • the single strand fraction is determined in the prior art by ion exchange or reverse phase HPLC.
  • this method is complicated, time-consuming and also requires a constant optimization of the running conditions.
  • pure samples of each single strand are needed as calibration standards.
  • the detection limit for the single-stranded part is around 5%. This can be explained inter alia by the fact that the constituents (strand A, B, or double strand) often have similar retention times in chromatography. An evaluation is thus by the "running together" Both components much more difficult and inaccurate.
  • no direct statements can be made on the single strand in excess (strand A or strand B) via HPLC. If both strands have no or too little retention difference in the HPLC method used, the determination of the single strand excess must be carried out using a further method.
  • PAGE polyacrylamide gel electrophoresis
  • MS mass spectrometry
  • mass spectrometry is often used to check the quality of synthetically produced nucleic acids after preparation.
  • a method known as LC-MS is known.
  • the liquid chromatography is coupled with mass spectrometry.
  • the liquid chromatography is used for separation and the mass spectrometry for the identification and / or quantification of the substances.
  • other detectors are switched on, such as UV-ELS or conductivity detectors.
  • MALDI-MS matrix assisted laser desorption ionization mass spectrometry
  • the invention relates to a method for determining at least one property parameter of a sample which contains at least one biomolecule, the method comprising the following steps:
  • step (c) preparing a mass spectrum of the sample containing the standard under denaturing conditions; (d) comparing the peak height or peak area of at least one biomolecule-allocatable peak in the mass spectrum of step (b) with the peak height of the corresponding peak in the mass spectrum of step (c).
  • a biomolecule is understood to mean a chemical compound which occurs naturally in living organisms or is or can be prepared by them.
  • Biomolecules consist mainly of carbon and hydrogen, along with nitrogen, oxygen, phosphorus, sulfur and other rarer elements.
  • the biomolecules consist of two non-covalently bound units, e.g. homo- or heterodimeric protein complexes or double-stranded DNA, the latter being preferred.
  • the biomolecule comprises at least one nucleic acid, in particular one or more oligomolecules, e.g. a mixture of single-stranded and / or double-stranded nucleic acids.
  • double-stranded part in the sample means the ratio of the amounts (molar amounts) of the part of a nucleic acid which is present as a double strand to that part of a nucleic acid which is present as a single strand.
  • the parts, which are present as double strand or single strand are each two complementary single strands, which together can form a double strand. It is thus possible for each of the complementary single strands, which are preferably not identical, to determine a double-stranded part or a ratio of the respective single strand to the double-stranded part in the sample.
  • nucleic acids contained in the sample DNA, RNA, non-natural derivatives thereof, such as LNA, and preferably natural or synthetically produced nucleic acids, so-called oligonucleotides, can be used.
  • the ratio of single strands to double strands in the sample, the excess of single strand in the sample, the interaction forces between different nucleic acids of the sample or the interaction of the in the Sample present nucleic acid with other non-nucleic acid-containing ingredients of the sample can be determined.
  • the method according to the invention is described by way of example for the measurement of nucleic acids, in particular double-stranded and single-stranded nucleic acids.
  • it may be suitably extended to other biomolecules, e.g. Proteins which are correspondingly transferred in complexes consisting of two non-covalently bound units, which would correspond to the double strand, and in corresponding monomers, which would correspond to the single strand.
  • corresponding refers to peaks which occur in several measurements and originate from the same substance.
  • corresponding peaks which are derived from a particular single strand, which under native conditions in the spectrum can only be seen at height, in which it is not bound in the double strand, and gives a stronger signal under denaturing conditions, since there is virtually no double strand is present.
  • the corresponding peaks are understood as meaning the respective peak pairs to be assigned to a particular single strand of two measurements, one of which is recorded under native and one under denaturing conditions.
  • normalization or “normalizing” is understood as meaning the multiplication of the peak heights / areas of one spectrum with respect to another by a factor which is determined so that a substance, preferably the standard, is kept constant in both measurements then having the same peak height or area in both spectra.
  • all measurements are normalized with respect to one measurement, in particular with respect to the standard held constant in the samples of all measurements.
  • the mass spectrum of a sample is obtained by measuring the sample in a mass spectrometer.
  • a mass spectrometer usable in the method of the present invention a MALDI-based mass spectrometer is preferred.
  • the sample is introduced into the instrument in the form of a so-called matrix.
  • the MALDI mass spectrometry is based primarily on the cocrystallization of matrix and analyte with a 100 to 100,000-fold molecular excess of matrix.
  • matrix substances small organic molecules are normally chosen which strongly absorb at the laser wavelength used (eg nitrogen lasers at a wavelength of 337 nm). With short high-energy laser pulses of a few nanoseconds pulse duration follows the excitation, which leads after relaxation in the crystal lattice to explosive particle separations on the surface of the matrix crystal. The combination with the matrix prevents fragmentation of mass-rich molecules.
  • the ionization mechanism in MALDI mass spectrometry is not fully understood.
  • the present invention is based, inter alia, on the problem that to quantify the content of double strand of a sample, e.g. consisting of two oligonucleotides, the signal intensities of the double and single strands in the MALDI mass spectrum can not be compared directly with one another, since the intensities obtained depend both on the mass of the detected nucleic acid and on the base composition itself.
  • a problem in the quantitative determination of the ratio of single strand to double strand and determination of the excess content on a single strand compared to the complementary single strand lies in the variation of the absolute values of the peak heights / peak areas of a mass spectrometric measurement to the next.
  • At least two measurements are carried out, one under native conditions in which the double strand is stabilized, and one under denaturing, the double strand destabilizing conditions.
  • An intensity comparison of the peaks of the corresponding single strands taking into account the internal standard provides a reliable statement about the single-stranded part of the sample.
  • an internal standard is each at two measurements are added and the single stranded intensity (Iss) relative to the intensity of internal standard (l S s) is determined. From the single-strand intensities single-strand and double-stranded parts can be calculated. Furthermore, it can be determined simultaneously which single strand is in excess, provided that the masses of the two strands differ (M Stran g A ⁇
  • an aqueous solution is first prepared consisting of the oligonucleotide probe and an added standard.
  • the standard e.g. comprises another oligonucleotide, must not interact with the sample under the experimental conditions, especially not under "native” and “denaturing” conditions.
  • the added standard acts as an internal standard.
  • a denaturing agent for example formic acid
  • the denaturing agent is weighted and added in such amounts (preferably 10-30% by weight, in particular 15-25% by weight, for example 20% by weight of the sample) that the double strand completely decomposes into single strands.
  • the measurement previously performed under "native" conditions is thus repeated under identical conditions with the mere addition of denaturing agent.
  • the intensity of the single strand that is not bound in the double strand is known (IssN at iv) - From the measurement under "denaturing” conditions is the intensity and thus the amount of the single strand contained in the sample known (Iss D e n a ⁇ - Taking into account the ratio factor y between the two different measurements of the proportion of the single strand can be determined therefrom at which it is present at “native” conditions (ISSN at iv (%)) • As this share with it If the proportion of single strand bound in double strand is 100% supplemented, this can be used to determine the proportion of single strand bound in double strand (I DS (%)).
  • the peak heights can be used in addition to the peak areas, provided that the standard and single strands are in a similar mass range, so that the resolution (peak width) is the same for both.
  • Suitable standard substances are all single-stranded oligonucleotides which show no interaction with the analyte substances under the experimental conditions and do not change under the influence of the denaturing agent.
  • the use of oligonucleotides also makes it easy to tune the mass range of the standard to the single strands, which, as explained above, leads to a comparable resolution.
  • a difference of 1 to 2 nucleotide units between the length of the oligonucleotides and the length of the individual strands to be examined is advantageous.
  • Such an analysis of the spectra obtained requires a linearity of the mass spectrometric measurement over two orders of magnitude of concentration, ie the concentrations of the single strands are between 1 and 100 percent of the measuring range of the mass spectrometer. It should also be noted that when measuring under "denaturing" conditions, the ratio of ion intensity of standard and analyte should not be affected compared to the measurement in "native" conditions to avoid falsification of the results of the
  • any excess of one of the two single strands (strand 1 or 2), the to form the double strand can be determined from the two measurements at "native" and “denaturing” conditions, any excess of one of the two single strands (strand 1 or 2), the to form the double strand.
  • the direct difference of the signal intensities of the two strands within a spectrum can not be used directly for this, since due to different ionization and detection probabilities of the two molecules, the excess is not directly, e.g. by subtracting the intensities.
  • the different ionization and detection probability of a molecule is taken into account by means of an unknown response factor z. Since the response factor z of one strand with respect to the other is between two measurements, e.g. under "native" and "denaturing" conditions, this unknown factor z can be eliminated by the ratio of the intensities between the measurements. In this way, the excess of one single strand over the other can be quantified.
  • the proposed double-strand quantification method according to the invention has the particular advantage that the peak ratios are determined in a single solution. By setting the intensities of the peaks of two measurements, fluctuations of the measurement parameters are eliminated and thus a high accuracy of the values to be determined is ensured.
  • the inventive method also has the advantages that the measurement is very fast due to the use of a MALDI mass spectrometer, since the method can be automated and the sample throughput is very high. Thus, typically only a few minutes are required to record the two measurements at "native” and “denaturing” conditions. The evaluation of the spectra and the corresponding calculations of the intensities and the excess ratio can easily be carried out with computer assistance. The use of calibration substances is not necessary, except for an internal standard, which can be the same every time. Thus, due to the above advantages, the error in determining the double strand portion can be suppressed to below 2 percent.
  • the inventive method can be carried out with any MALDI mass spectrometer, as long as it is a linear MALDI mass spectrometer.
  • the MALDI sample matrices useful in the method of the present invention are neutral matrices that do not result in denaturation of the duplexes. Particularly advantageous are saturated solutions of 6-aza-2-thiothymine (ATT, available from Fluka, Germany) (about 6-7 mg / ml) in 100-200 mM, particularly advantageously 120-150 mM, aqueous diammonium hydrogen citrate. Solution (DAHC, available from Fluka, Germany). With the concentration of DAHC increasing from 0 mM, the concentration of detectable duplex increases and approaches saturation at 150-200 mM (see Example 2).
  • ATT 6-aza-2-thiothymine
  • DAHC available from Fluka, Germany
  • the method according to the invention is particularly advantageous given a length of the nucleic acids of 10 to 60 bp, in particular of 18 to 23 bp, e.g. of siRNA.
  • a lower limit for sufficient stability of the duplex should be around 10 bp.
  • the invention further relates to the use of an acid for denaturing nucleic acids in sample preparation for mass spectrometry.
  • the invention relates to the use of a mixture of 6-aza-2-thiothymine (ATT) and diammonium hydrogen citrate (DAHC) in a sample matrix for MALDI mass spectrometry.
  • ATT 6-aza-2-thiothymine
  • DAHC diammonium hydrogen citrate
  • the method according to the invention is also suitable for the detection of other, non-covalent interactions between nucleic acid single strands which have complementary base sequences.
  • the relationship between the species present as a single strand and as a double strand on the binding energy for the formation of the double strand from the respective strands can be deduced.
  • Appropriate method for Determination of the binding energies from the binding ratios are known in the art.
  • the method according to the invention can be used to determine protein interactions since, in accordance with the above statements, the ratio between dissociated proteins and protein complexes can be determined in an identical manner. From the ratio determined by means of the method according to the invention, conclusions can be drawn about corresponding interaction energies.
  • the method of the invention can be used to control the quality of synthetically produced, double-stranded oligonucleotides, as described, for example, in US Pat. used in gene expression studies (siRNA or RNAi).
  • the process according to the invention can be advantageously used for the preparation of mixtures with a defined ratio between double strand and single strand. This is e.g. possible via mixture titration with subsequent MALDI mass spectrometry according to the invention.
  • a simple method is thus provided to obtain compounds containing a defined amount of duplex, preferably mainly duplex, e.g. > 98%, exist. Such compounds are particularly important for pharmaceutical applications of great importance.
  • FIG. 1 shows a graph with two offset mass spectra of a sample in native and denaturing conditions.
  • FIG. 2 shows a diagram in which the double-strand / single-strand intensity of a nucleic acid-containing sample is plotted against the DAHC concentration of the sample solution.
  • example 1 The invention is further illustrated by the following examples: example 1
  • Figure 1 shows two staggered MALDI-MS spectra, where lane 1 was recorded under "native" conditions and lane 2 under “denaturing” conditions. Only the mass range of the standard and the single strands are shown since the peaks of the double strand are not needed for the evaluation.
  • the standard used was an oligonucleotide strand which does not interact with the complementary single strands 1 and 2 (SS 1 and SS 2 ) to be investigated. Since the standard in the samples of the two measurements is identical, the intensities of the two spectra can be normalized via the correction factor y, which is determined according to formula (1) above, so that subsequently the intensity of the standard is the same in both spectra and respective single-strand intensities are thus directly comparable. For the spectrum shown in FIG.
  • a possible excess of a single strand can be determined.
  • the values of single strand 2 (SS 2 ) of the peaks at "native" conditions (1347 counts) and at "denaturing" conditions (16749 counts) are required. Used in the above formula (4b) thus results in a relative excess for single strand 2 of 2.5%. That is, single strand 1 is a total of 5.7% in the form of single strand and single strand 2 8.2% in the form of single strand, with single strand 2 has a relative excess of 2.5%.
  • RNA 6-aza-2-thiothymine
  • DAHC aqueous diammonium hydrogen citrate solution
  • RNA oligonucleotide was 17-base-pair RNA oligonucleotide that did not interact with the sample under the experimental conditions.
  • Mass spectrometric analysis was performed on a linear time MALDI time-of-flight mass spectrometer (Voyager De-Pro, Applied Biosystems, Framingham, USA). Spectra of the positive ions as well as the negative ions can be used for analysis since both show comparable intensities and resolutions.
  • the operating parameters chosen were an acceleration voltage of 20 kV, a grid voltage of 95% of the acceleration voltage and a delay time of 600 ns. In order to obtain a sufficient signal-to-noise ratio, 50-100 individual spectra were accumulated for each spectrum. To calculate the peak areas and heights, the spectra were smoothed 19 pt and the baseline corrected.
  • the concentration of double strand was found to increase with increasing DAHC concentration, approaching saturation at 150-200 mM.
  • the samples were prepared and measured according to Example 1.
  • the ratios of double strand to single strand were calculated according to the above-mentioned method of the measured according to Example 1 spectra.

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  • Chemical & Material Sciences (AREA)
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Abstract

L'invention concerne un procédé spectrométrique de masse pour la détection et la quantification d'acides nucléiques à double brin qui sont associés de manière non covalente.
EP07856466A 2006-12-08 2007-12-07 Procédé spectrométrique de masse sur des échantillons contenant des acides nucléiques Withdrawn EP2097537A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EP07856466A EP2097537A1 (fr) 2006-12-08 2007-12-07 Procédé spectrométrique de masse sur des échantillons contenant des acides nucléiques

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
EP06025448A EP1930444A1 (fr) 2006-12-08 2006-12-08 Procédé spectrométrique de masse sur des échantillons contenant des acides nucléiques
PCT/EP2007/010663 WO2008068034A1 (fr) 2006-12-08 2007-12-07 Procédé spectrométrique de masse sur des échantillons contenant des acides nucléiques
EP07856466A EP2097537A1 (fr) 2006-12-08 2007-12-07 Procédé spectrométrique de masse sur des échantillons contenant des acides nucléiques

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EP2097537A1 true EP2097537A1 (fr) 2009-09-09

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EP07856466A Withdrawn EP2097537A1 (fr) 2006-12-08 2007-12-07 Procédé spectrométrique de masse sur des échantillons contenant des acides nucléiques

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JP5072682B2 (ja) * 2008-03-28 2012-11-14 富士フイルム株式会社 質量分析用デバイス、これを用いる質量分析装置および質量分析方法
AU2014223472B2 (en) 2013-02-28 2018-03-08 Nuvera Fuel Cells, LLC Electrochemical cell having a cascade seal configuration and hydrogen reclamation
CN119757444B (zh) * 2024-12-19 2025-09-23 中国科学院精密测量科学与技术创新研究院 一种基于核磁共振磷谱鉴别游离核酸与被载体包裹的核酸的方法

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US6723564B2 (en) * 1998-05-07 2004-04-20 Sequenom, Inc. IR MALDI mass spectrometry of nucleic acids using liquid matrices
EP2186879B1 (fr) * 2002-10-03 2016-11-23 Norman Leigh Anderson Quantification à sensibilité élevée de peptides par spectrométrie de masse
WO2006015797A2 (fr) * 2004-08-09 2006-02-16 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. Procédé de criblage de rendement élevé de liaison de molécules de test avec des molécules cibles

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DATABASE MEDLINE [online] US NATIONAL LIBRARY OF MEDICINE (NLM), BETHESDA, MD, US; December 2004 (2004-12-01), SAKAI H ET AL: "Simultaneous detection of Staphylococcus aureus and coagulase-negative staphylococci in positive blood cultures by real-time PCR with two fluorescence resonance energy transfer probe sets.", Database accession no. NLM15583307 *
JOURNAL OF CLINICAL MICROBIOLOGY DEC 2004 LNKD- PUBMED:15583307, vol. 42, no. 12, December 2004 (2004-12-01), pages 5739 - 5744, ISSN: 0095-1137 *
KURIHARA K ET AL: "The hyperchromic effect of organic denaturing reagents on deoxyribonucleic acid", BIOCHIMICA ET BIOPHYSICA ACTA, ELSEVIER, NL LNKD- DOI:10.1016/0006-3002(63)90165-3, vol. 68, 1 January 1963 (1963-01-01), pages 434 - 445, XP023380476, ISSN: 0006-3002, [retrieved on 19630101] *
See also references of WO2008068034A1 *

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WO2008068034A1 (fr) 2008-06-12
US20100167410A1 (en) 2010-07-01
EP1930444A1 (fr) 2008-06-11

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