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WO2004106545A1 - Procedes permettant une detection amelioree au moyen de techniques sensibles a la surface - Google Patents

Procedes permettant une detection amelioree au moyen de techniques sensibles a la surface Download PDF

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Publication number
WO2004106545A1
WO2004106545A1 PCT/EP2004/050842 EP2004050842W WO2004106545A1 WO 2004106545 A1 WO2004106545 A1 WO 2004106545A1 EP 2004050842 W EP2004050842 W EP 2004050842W WO 2004106545 A1 WO2004106545 A1 WO 2004106545A1
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Prior art keywords
template
analyte
polymensation
specific
degradation
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PCT/EP2004/050842
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English (en)
Inventor
Michael Wijnhoven
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Fujirebio Europe NV SA
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Innogenetics NV SA
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    • 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
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/682—Signal 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
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/6825—Nucleic acid detection involving sensors

Definitions

  • Another aim ot the invention is to provide a method for continuous measurement of binding, hybndisation, dissociation and similar processes by means of a surface sensitive technique
  • the present invention provides methods for the detection of the presence of an analyte using template independent polymensation and or template selective degradation, the products of which are measured as a discrete change in surface excess using a surface sensitive technique
  • Detection of the analyte is achieved by means of addressing suitable initiation sites for template independent polymensation present within the analyte or associated with the analyte Henceforth analyte specific template independent polymensation by means of suitable enzymes such as glycosyltransferases and nucleotidylexotransferases can be used to change the surface excess as measured using a surface sensitive technique
  • the present invention further provides a functional signal amplification by means of analyte specific template independent polymensation, wherein the surface excess may be continuously measured, for example as a function of a physical or chemical gradient It is a specific advantage that such functional amplification allows for charactensation of the interaction between analyte and discrete specific affinity sites In practice this makes it possible to positively discnminate mismatches in nucleic acid sequences, or detect multiple interactions between analyte and affinity sites, without the requirement for any further steps
  • the present invention provides methods for the detection of an analyte using template selective degradation To this extent discrete specific affinity sites containing degradation precursors are degraded in presence of the analyte and a suitable agent catalysing template selective degradation The presence of the analyte is then detected by measunng a change in surface excess at a transducer surface Alternatively, the degradation precursors contain in addition also initiation precursors for template independent polymensation which are activated by said template selective degradation
  • template independent polymensation and template dependent degradation may be combined to detect the presence of an analyte by measurement of the surface excess
  • template selective degradation may be used to create an initiation site for template independent polymensation
  • the present invention further includes methods for the analysis of nucleotide sequences using the advantages of functional signal amplification, and it's measurement by means of a surface sensitive technique
  • FIG IA Cyclic voltammogram of a hyb ⁇ dization using a complementary oligonucleotide 1) before hybndisation 2) after hybndisation and template independent polymensation
  • FIG I B Cyclic voltammogram of a hybndisation using a non complementary oligonucleotide I) before hybndisation 2) after hybndisation and template independent polymensation
  • FIG 1C Cyclic voltammogram of a hybndisation using a complementary PCR product (sample I) 1) before hybndisation, 2) after hybndisation, 3) after hybndisation and template independent polymensation
  • FIG 2B Temperature jump expenment showing the course of denaturation of electrodes hyb ⁇ dized with respectively complementary oligonucleotide and a oligonucleotide sequence with a transition mismatch and amplified using DNA nucleotidylexotransferase 0 represents the relative surface coverage
  • FIG 3A Differential dissociation plot of homogeneous and heterogeneous surfaces, obtained from equilibnum dissociation using cyclic voltammetry Homogeneous surface 25-mer (Senes 1), Heterogeneous surface containing a 25-mer and 18-mcr complementary oligonucleotide (Senes 2), Homogeneous surface containing a 18-mer complementary ol ⁇ gonucleot ⁇ de(Se ⁇ es 3)
  • FIG 3B Difference differential dissociation plot of a heterogeneous surface containing 18 and 25-mer complement Senes 1 25-mer homogeneous surface subtracted, Senes 2 18
  • FIG 4 Temperature jump expenment analyzing the outcome of a restncted length polymenzation expenment
  • the parameter ⁇ represents the relative surface coverage Senes 1 and 2 ACG extension, senes 3 and 4 ACT extension (for details see example IV)
  • FIG 5 Detection of specific endonucieolytic activity of RNase H (duplicates) (1) Chime ⁇ c probe and complementary template, (2) Chimenc probe and partially complementary template, (3) Chime ⁇ c probe and non complementary template, (4) chime ⁇ c probe, no template, (5) chime ⁇ c probe and complementary template no RNase H, (6) buffer only (for details see example V)
  • Qi and Q respectively represent the integrated surface charge before and after the expenment
  • FIG 6 Real time monitoring of DNase I induced degradation of a transducer surface containing specific affinity sites previously hybndised with complementary DNA ohgonucleotides and subjected to template independent polymerization Senes 1 No DNase added, Senes 2 DNase added
  • analyte or specific analyte is used to desc ⁇ be a substance which is the object of detection and further contains, or is made to contain an initiation site or initiation precursor for template independent polymensation Alternatively the term is used to desc ⁇ be an analyte which contains a precursor for template dependent degradation
  • analyte is also used to specify those substances which are denved from the analyte by means of a specific process, including particular reaction products further defined below
  • nucleic acid sample refers to a sample containing nucleic acids, which may have been subjected to a suitable punfication or extraction method known in the general art of biochemistry The term is also more widely used to designate the analyte or sample to be investigated further dcscnbcd by the terms "specific reaction product” and "de ⁇ vative thereof
  • specific reaction product and “denvative thereof refers to the products obtained in a specific reaction with respect to nucleic acids It compnses but is not limited to the products obtained from a specific amplification reaction (e g polymerase chain reaction, ligase chain reaction, transcnption mediated amplification, cycling probe amplification etc ), a nucleic acid hyb ⁇ dization reaction to probes of known o ⁇ gin, or a reaction involving the enzymatic redist ⁇ bution of nucleic acid sequence information due to for example the activity of specific nucleases, specific chemical cleavages, or polymerase based sequencing reactions
  • the tenn specific reaction product also encompasses reactions relating to the combination of specific reactions such as a polymerase based amplification used in conjunction with a specific nuclease reaction or sequencing reaction
  • the specific reaction product further may include reactions involving the deprotection of soluble nucleic acid probes by means of the specific activity of a DNA or RNA endo- or
  • initiation site is used to desc ⁇ be a specific site corresponding to the substrate to which monomers may be added or removed in a template independent or template dependent manner
  • template dependent polymensation refers to the stepwise addition of monomer units to an initiation site (also referred to as a primer), each of them directed by the properties of a template
  • initiation precursor is used to descnbe the initiation site which needs activation m order to become a substrate for template dependent or independent polymenzation or degradation, and preferably due to a specific event relating to the nature and composition of the said initiation precursor
  • degradation precursor is used to desc ⁇ be the particular entity that is prone to a degradation process This entity is contained within the analyte, results from binding of analyte to affinity sites, or is contained within the affinity sites but only activated upon binding of the analyte
  • template independent polymenzation refers to the stepwise or processive addition of monomer units to the initiation site without the requirement for a template
  • the term "functional amplification” refers to the creation of a signal amplification which retains some of the properties of the amplified target
  • structural amplification refers to an amplification of a specific reaction product or sample which retains some of the properties of the amplified target which by amplification induces a significant change in molecular composition, thereby forming the basis for detection of a specific sample or reaction product In the present context it is used to descnbe a change in the gross macromolecular composition of a transducer surface
  • surface excess is used to descnbe a particular surface composition, such as the pnmary excess of a polyanion, polycation or other relevant species and components associated to them and implies that a difference exists at the interface between transducer surface and bulk medium
  • surface sensitive techniques applies to physical methods that allow one to characte ⁇ ze or measure surface compositions and changes thereof, including but not limited to faradayic and lmpedunetnc techniques, which are sensitive to charge transfer, conductivity and dielectnc phenomena These methods may typically combine chemical specificity and features relating to the overall composition of a surface Mass sensitive methods which include but are not limited to resonance methods such as the quartz crystal balance, optical wave guide methods for example resonant mirror and surface plasmon resonance in which the surface itself is transducer and work function based techniques such as the Kelvin microprobe are also included It should be clear that the list of surface sensitive techniques is bound to be incomplete, and several new techniques are being developed
  • transducer surface relates to the integrated assembly of a surface which may be used in conjunction with one of the above mentioned surface sensitive techniques
  • the transducer surface in general may be part of a passive transducer (e g piezoelectric transducers, optical transducers, mechanical transducers etc ) or an active transducer (e g resistance or reactance transducers) or hybnd devices that are classified according to their stimulus requirements
  • modified electrode and more general “modified transducer surface” is used to specify chemically modified electrode surfaces having specificity for a specific nucleic acid sequence or proteins, or in general have a selective affinity for a particular analyte or improve the resolution of its detection
  • modification may consist of nucleic acid sequences, or nucleic acid analogs which arc not found in nature
  • Electrode surfaces may thus be modified according to the nature of the electrode matenal
  • Several examples relating to the controlled modification, functionahsation of electrode surfaces as well as the general background relating to the art are descnbed in "Integrated chemical systems” by Allen J Bard (A J Bard, Wiley-Interscience 1994)
  • specific affinity site is used to desc ⁇ be a molecule or molecular property which has been conferred to an otherwise non-specific earner, thereby inducing the propensity to specifically associate with a closely defined range of molecules under particular
  • pen-electrodic space is used to specify the space in the immediate vicinity of the electrode, the latter being defined as a conducting substance having a connection to the external measunng circuitry Apart from the solvent or electrolyte in the immediate vicinity of the electrode surface, a regular reproducible structure having different properties with respect to conductivity and or capacitance may be included into the space defining the bounda ⁇ es of a particular measurement area
  • pen-electrodic space may thus be applied to both intra and inter electrode configurations, depending on whether this space involves single or multiple distinct connections to the measunng circuitry
  • fibrous carbon electrodes consisting of both conductive and non conducting compounds allowing surface modification as desc ⁇ bed in "Chemically modified carbon fibers"(I N Ermolenko et al Eds , VCH 1990)
  • fibrous carbon electrodes consisting of both conductive and non conducting compounds allowing surface modification as desc ⁇ bed in "Chemically modified carbon fibers"(I N Ermolenko et al Eds , VCH 1990)
  • exogenous label is used to specify a chemical entity which has a chemical or physical property distinct from the sample molecule to be detected and allows to detect the sample molecule
  • exogenous labels include radioactive, fluorescent, or enzyme binding sites which may become associated with the sample by means of a probe or are integrated in the probe
  • the general concept behind the present invention relates directly to the use of surface sensitive techniques These techniques allow for the measurement of any substance, without the need for labels, to give nse in a measurable signal by assessing parameters that are directly altered by the presence of said substance It has been found to be very advantageous to exploit this even further by using the presence of said substance as a prerequisite step to magnify the signal, which results from said substance, even further It has been found that such can be achieved in an analyte specific way by using processes that draw upon the presence of said analyte to either magnify the signal by building more substance or by doing the reverse, and destroying the signal that was already generated by the presence of said analyte
  • the first can be achieved by template independent polymensation provided that the analyte present contains an initiation site for said template independent polymerisation
  • the second can be achieved by template selective degradation provided that the analyte present contains an initiation site for said template selective degradation
  • the present invention provides a method for detecting the presence of an analyte in a sample by means of modifying and measunng the surface excess wherein the said method compnses a) increasing surface excess by means of template independent polymerization or b) decreasing the surface excess by means of template selective degradation
  • the present invention provides a method for detecting the presence of an analyte in a sample compnsing the following steps a) Reacting the sample with a reaction mixture that causes template independent polymensation, by means of addressing suitable initiation sites present within, or specifically associated with, the analyte b) Assessmg the template independent polymensation, by means of measuring the surface excess using a surface sensitive technique
  • the present invention also provides a method for detecting the presence of an analyte in a sample compnsing the following steps a) Binding the analyte to a surface containing discrete specific affinity sites that are selective for the said analyte b
  • the current mvention thus provides a method for detecting specific analytes (containing initiation sites for template independent polymensation) by means of a transducer surface and template independent polymerization or template selective degradation
  • the transducer surface may for this purpose either contain, be brought in the vicinity of, or in contact with the analyte in order to measure the surface excess at the said surface
  • a tangible interaction between transducer surface and analyte as defined by the surface excess is understood to be present
  • the signal is amplified in a analyte specific way by means of template independent polymensation of suitable initiation sites contained within the said analyte
  • Such initiation sites may be present within the specific analyte (being thus a natural property or intrinsic part of the analyte), or associated with the analyte by means of an analyte specific process which couples a natural or synthetic initiation site to the
  • the said analyte interacting with the transducer surface is a nucleic acid sample, a specific reaction product or denvative thereof
  • the said analyte may be detected by template independent polymenzation of the initiation sites present within or associated with the said nucleic acid sample
  • initiation sites for template independent polymensation may for example be the 3'OH termini which are contained within the sample, and are readily amenable to the addition of nucleoside residues by a template independent polymensation process involving a nucleotidylexotransferase Consequently a sample containing a nbonucleic or deoxynbonucleic homo or copolymer is synthesized, this structural amplification or increase may then be detected at the transducer surface
  • Suitable enzymes capable of performing template independent polymensation may be employed to detect samples containing DNA, RNA or both at a transduc
  • initiation sites for template independent polymensation may be specifically associated with the said nucleic acid sample, specific reaction product or denvative thereof This may for example compnse the hybndisation of suitable initiation sites to the said sample, by means of exploiting the propensity of nucleic acids to form stable and specific hydrogen bonds with ohgonucleotides of complementary or partially complementary sequences
  • ohgonucleotides may contain initiation sites for template independent polymensation, or have to them attached initiation sites for template independent polymensation tor example by means of covalent modification, further hybndisation, or enzymatic modification in a analyte specific way
  • initiation sites may as before consist of nucleoside residues, and subjected to template independent polymerisation by means of using a suitable nucleotidylexotransferase, in order to detect the said sample at the transducer surface It will be obvious, that multiple initiation sites may be attached to the analyte by exploiting sequence
  • the specific analyte may be a protein, carbohydrate, a collection thereof, or a specific reaction product or de ⁇ vative of said analyte, which contains initiation sites for template independent polymensation
  • initiation sites for example may be naturally present on specific types of proteins such as glycoproteins as a result of post translational modification, or on proteoglycans, the latter being more complex structures
  • Oligo or polysacchandes present on such specific proteins may hence be used as initiation sites for template independent polymensation by means of a suitable hexosyltransferases (or combination thereof), or pentosyltransferases in order to detect a change in surface excess at a transducer surface in an analyte specific way
  • high mannose glycoproteins may be detected using specific glycosyltransferases capable of utilising mannose residues as an initiation site for template independent polymensatioa
  • proteins having a natural tendency to associate with nucleic acid sequences the latter being initiation sites for
  • the specific analyte is a protein, carbohydrate,a collection thereof, or a specific reaction product or denvative of said analyte which may be treated to contain associated initiation sites for template independent polymensation in order to measure a change in surface excess at the transducer surface in a analyte specific way
  • ohgosacchande, or oligonucleotide initiation sites may be_assoc ⁇ ated with the analyte of interest or to another protein or peptide having a specific affinity for the said protein sample (e g reporter)
  • suitable ohgosacchande initiation sites may be directly associated with the analyte
  • this may be achieved by means of forming an ⁇ -l ⁇ nkage to accessible senne or threonine residues, a reaction which may be catalysed by O-N-acetyl-D-glucosamine transferase (EC 2 5 1 7), or using specific lectin
  • the nucleic acid sample may be immobilized to the said surface by means of incorporating specific chemical functionalities mto the sample allowing a stable interaction with this surface
  • funchonalisation may involve the incorporation ot biotin or modified nucleotides such as phosphorothioates dunng nucleic acid amplification
  • said nucleic acid sample or specific reaction product may be immobilised by means of oligonucleotide sequences which are complementary to specific oligonucleotide probes, or suitable affinity sites immobilised at the said surface at one end, and have a sample specific sequence at the other end
  • the analyte specific detection by means of template independent polymensation provides a functional signal amplification and allows the continuous measurement thereof at a transducer surface
  • products formed according to the methods described in the present invention may be further analysed, by subjecting the product of template independent polymensation to conditions involving temperature, hydrophobicity, ionic strength and so on, while measunng the surface excess as a function of the applied conditions
  • the ⁇ nteract ⁇ on(s) between the analyte and discrete specific affinity sites used to immobilise the said analyte, as well as ⁇ nteract ⁇ on(s) between analyte and associated initiation precursors may be charactensed (subsequent to template independent polymensation)
  • both kinetic and equilibnum mfo ⁇ nation relating to the said interactions may be collected by respectively performing temperature jump or equilibnum dissociation exp
  • further information regarding the nature of the analyte may be obtained by further subjecting the products obtained using template independent polymensation to template selective degradation
  • template selective degradation For this purpose specific DNA or RNA exo or endo-nucleases, specific endo or exoglycosidases, or other specific biological or chemical agents may be used This for example allows one to investigate the interaction of a specific affinity site such as a oligonucleotide probe and a nucleic acid sample by means of a selective degradation agent such as a restnction endonuclcasc Or in the case the analyte is a carbohydrate the initiation sites used for template independent polymensation may be cleaved off using a specific endo- glycosidasc or glycanasc
  • the present invention provides a method for detecting the presence of an analyte in a sample compnsing a) Reacting the sample with a reaction mixture that causes template selective degradation, by means of addressing suitable degradation precursors
  • initiation sites for template independent polymensation may be produced in a analyte specific manner by means of template selective degradation of soluble or immobilised specific affinity sites interacting with the said analyte
  • template dependent degradation or cleavage of the specific affinity sites interacting with said analyte may be achieved according to the methods previously desc ⁇ bed in the current invention (previous embodiment)
  • immobilised specific affinity sites which are cleaved or degraded upon specific interaction with the analyte and if necessary auxiliary ohgonucleotides
  • suitable termini which may serve as initiation sites for template independent polymensation
  • specific affinity sites having 3' termini which are not capable to serve as initiation sites for template independent polymensation may be modified by means of analyte specific clea
  • the analyte is a nucleic acid sample, specific reaction product or a de ⁇ vative thereof which may be detected by measunng the surface excess at a transducer surface, wherein said surface excess is produced by a process involving template independent polymensation in conjunction with template selective degradation
  • the analyte is a nbonucleic acid
  • said analyte may be hyb ⁇ dised to a sequence specific affinity site at a surface and washe Following this step the hyb ⁇ dised nbonucleic acid is subjected to a reaction mixture containing polynucleotide adenylyltransferase and nbonuclease IV, the latter of which cleaves the product of template independent polymensation to yield new initiation sites for template independent polymensation
  • the product of the reaction may be detected by means of a surface sensitive technique at a surface containing specific affinity sites for the product of template independent polymensation
  • Gold electrode matenal either evaporated on silicon wafers or polymer earners were obtained from both commercial and non-commercial sources
  • the electrodes were mounted on glass slides or plastic sheet matenal using a low temperature curable epoxy (Epotek H-54), electncal connections between the electrode matenal and the electncal winng were established using silver loaded epoxy and graphite ink, the structure was then thoroughly sealed with epoxy leaving only the desired electrode matenal exposed
  • Structures for impedimet ⁇ c sensing involving a lnterdigitated electrode architecture were obtained from Imec (Belgium), or commercial suppliers, with lnterelectrode spacings varying between 1 and 40 ⁇
  • the gold electrode surfaces were subsequently inspected and activated by potentiodynamic cycling in a three electrode cell between -0 3 Volt and 1 2 Volt (versus Hg HgS ⁇ 4 reference) until steady state voltammograms were obtained Cyclic voltammograms and other electrochemical measurements were collected using a EGG PAR 283 pot
  • Oligonucleotide probes and synthetic complements were purchased from Eurogentec (Belgium) and used without further pu ⁇ fication All chemicals used were analytical grade or better, CofPhen) 3 + 3+ was prepared according to the method as desc ⁇ bed by Dollnnore and Gillard (Dollimore, J Chem Soc Dalton, 1973, 933- 940 ) and modifications thereof Modification of the gold electrodes was earned out taking advantage of the well known gold sulfur interaction, which will be readily apparent to the person skilled in the art B ⁇ efly 3' or 5' thiol labelled ohgonucleotides are dissolved in a high salt acidic buffer (e g 1M KII 2 P ⁇ pH 4 5) to a final concentration of 3 ⁇ M, further containing 2 ⁇ M 3-mercaptopro ⁇ ylmethyld ⁇ methoxys ⁇ lane (ABCR, Germany) Freshly cleaned electrode surfaces are then fully covered with the modification solution, and left to react for 90 minutes at room temperature Subsequently the electrode surfaces are
  • Sequence selective electrode surfaces, or impedimetnc sensing structures using nucleic acid probes with a 3' modification group such as a thiol are prepared according to the procedure as descnbed in mate ⁇ als and methods section
  • the response of the modified electrode is checked and hybndised with a sample nucleic acid for example, a oligonucleotide complement (25-mer complementary sequence (5 nM) and 18-mer non complementary sequence (10 nM) F ⁇ g I A, or 18-mer non complementary sequence (10 nM) alone Fig 1 B), or a polymerase chain reaction product (sample 1 Fig 1 C, sample 2 Fig 1 D), in a suitable hybndisation solution (e g 200mM Na 2 S0 4 and 10 mM Tns-HCl pH 8 5) for about 60
  • the target was amplified at Innogenetics Research Labs according to methods known to the art, for convenience one phosphorylated pnmer was used allowing the generation of single stranded amphcon by means of phage lambda exonuclease Targets further are used as received and diluted in the hybndisation buffer (1/10) Different samples (sample 1 and sample 2) are used corresponding to vanations in the target sequence, and measured with respect to the nucleic acid probe at the electrode surface
  • Sequence selective electrodes (using a 25-mer oligonucleotide having a 3'- thiol moiety) are prepared as descnbed in the matenal and methods section Following modification the surfaces are hyb ⁇ dised under low stnngency conditions with sample ohgonucleotides which are either fully or partially complementary to the sequence immobilized at the electrode surface (see below)
  • the electrodes are subsequently subjected to template independent polymensation by DNA nucleotidylexotransferase usmg thymidme residues
  • the electrodes are analyzed using either the equilibrium dissociation or temperature jump method
  • the response of the electrodes to thermal equilibnum dissociation (figure 2A) or a temperature jump (at 52 8°C) (figure 2B) are reconstructed from the integrated cu ⁇ ents as obtained by continuous cyclic voltammetry
  • Sequence selective electrode surfaces are prepared as desc ⁇ bed in the matenals and methods section The response of the electrode is checked for modification, and challenged with sample or a specific nucleic acid reaction product
  • electrode surfaces are incubated with respectively 1 -mer and 25-mer complementary ohgonucleotides and mixtures thereof Following hybndisation, the electrode surface is subjected to template independent polymensation using DNA nucleotidylexotransferase Subsequently a temperature gradient (for equilibnum dissociation, see Fig 3A and 3B) or jump is applied to the transducer surface, while the surface excess is continuously monitored using for example cyclic voltammetry Sample ohgonucleotides used
  • Nucleic acid probe modified electrode surfaces are challenged with a nucleic acid pnmer which satisfies particular pruning cntena known by those skilled in the art
  • Complementary nucleotides are incorporated using a polymerase such as DNA polymerase I klenow fragment in presence of a rest ⁇ cting nucleotide mixture (0 ImM each dN 1 P, 004 U ⁇ l ' Klenow fragment, 50 mM I ns-HCl pH 7 5, 1 OniM MgCl 2 )
  • a polymerase such as DNA polymerase I klenow fragment
  • a rest ⁇ cting nucleotide mixture 0.
  • 3 of the four nucleotides are used, and as such polymenzation is terminated when no matching nucleotide is present
  • the extension reaction is earned out in solution in which a nucleic acid sample is present, and the extended pnmers are hybndized to the electrode surface Following extension and hyb
  • sample sequences are incubated in conjunction with chime ⁇ c probes and RNase H B ⁇ efly 0,1 ⁇ M sample sequence and 0,5 ⁇ M chimenc probe are incubated in plastic tubes in the presence of RNase H in a buffer (e g T ⁇ s HC1 pH 7,5) containing 8 to 10 mM MgCl 2 at
  • Sequence selective electrode surfaces having 3'th ⁇ ol moiety are prepared as desc ⁇ bed in the mate ⁇ als and methods section Subsequently complementary deoxyohgonucleotide is immobilized to the electrode surfaces and the obtained surface is subjected to template mdependent polymenzation using DNA nucleotidylexotransferase
  • the obtamed electrode surface may now be examined for the activity of a particular nuclease
  • the electrode is incubated with DNase I (2 Units / 100 ⁇ l), in a buffer containing 10 mM MgCI 2 , 50 mM Tns-HCl pH 76 and 5 mM Fe(CN)6 3 '* l" e electrode surface is subsequently continuously cycled in a conventional 3 electrode cell between 05 V and -07 V versus Ag/AgCI not compensated for IR drop thermostattcd at 37°C (see Fig 6)

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Abstract

L'invention concerne de procédés pour détecter et analyser des analytes spécifiques, au moyen de techniques sensibles aux excès de surfaces. La détection d'analytes tels que des acides nucléiques et d'autres biopolymères est effectuée au moyen de la polymérisation indépendante de gabarits, ou par dégradation sélective. Grâce à des sites d'initiation spécifiques d'analytes pour une polymérisation indépendante de gabarits et leurs mesures au moyen d'une technique sensible à la surface, un procédé d'amplification des signaux est mis en place. Lorsque ce dernier est combiné avec des sites d'affinité spécifiques immobilisés pour l'analyte, des informations détaillées par rapport à l'interaction entre l'analyte et les sites d'affinité spécifiques peuvent être obtenues. Des applications focalisent sur la détection d'un acide nucléique sur la base de réactions et l'utilisation desdits procédés permet de caractériser les analytes d'acides nucléiques. L'invention concerne, de plus, des procédés pour surveiller en temps réel des substrats d'acide nucléique sur la base de l'activité enzymatique sur la surface. L'invention concerne, en outre, des exemples utilisant des techniques voltamétriques et impédancemétriques.
PCT/EP2004/050842 2003-05-28 2004-05-18 Procedes permettant une detection amelioree au moyen de techniques sensibles a la surface Ceased WO2004106545A1 (fr)

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Publication number Priority date Publication date Assignee Title
WO1990013666A1 (fr) * 1989-05-11 1990-11-15 Amersham International Plc Methode de mise en sequence
WO1997004129A1 (fr) * 1995-07-14 1997-02-06 Biacore Ab Procede de sequençage de l'acide nucleique
WO2001031057A2 (fr) * 1999-10-22 2001-05-03 Nanogen Recognomics Gmbh Sondes pour acide nucleique a brin double et leur utilisation

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