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EP2250284A1 - Microréseau pour l'analyse de séquences nucléotidiques - Google Patents

Microréseau pour l'analyse de séquences nucléotidiques

Info

Publication number
EP2250284A1
EP2250284A1 EP09709868A EP09709868A EP2250284A1 EP 2250284 A1 EP2250284 A1 EP 2250284A1 EP 09709868 A EP09709868 A EP 09709868A EP 09709868 A EP09709868 A EP 09709868A EP 2250284 A1 EP2250284 A1 EP 2250284A1
Authority
EP
European Patent Office
Prior art keywords
sequence
ligating
segment
labelling
microarray
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Withdrawn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
EP09709868A
Other languages
German (de)
English (en)
Inventor
Harry Benjamin Larman
Andrea Cuppoletti
Barbara Pisanelli
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.)
TWOF Inc
Original Assignee
TWOF Inc
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 TWOF Inc filed Critical TWOF Inc
Publication of EP2250284A1 publication Critical patent/EP2250284A1/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Withdrawn legal-status Critical Current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
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
    • C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips

Definitions

  • the present invention relates to a microarray for the analysis of nucleotide sequences.
  • probe strand indicates an oligonucleotide which is covalently or non-covalently attached to a substrate for the formation of microarrays and comprises a probe sequence which is complementary to a specific nucleotide sequence to be analysed.
  • the expression “labelling segment” indicates an oligonucleotide, to which a fluorophore moiety is bound and which is adapted to hybridise to a complementary sequence of the probe strand.
  • ligating segment indicates an oligonucleotide adapted to hybridise to a complementary sequence of the probe strand and to ligate an end thereof to a nucleotide sequence hybridised or partially hybridised to the probe strand through a ligating step .
  • probe hybrid indicates a complex formed by a probe strand and at least one labelling sequence hybridised one to the other.
  • template strand indicates a nucleotide strand used to generate a probe strand.
  • the microarray formed by immobilised template strands is defined as microarray template.
  • SuNS is a molecular stamping technique for generating microarrays, which is disclosed and claimed in PCT patent application WO2006112815 herein incorporated by reference.
  • the method consists in the generation of nucleotide strands by replicating template strands, and in the subsequent stamping of these molecules on a new substrate by means of the SuNS technique.
  • MicroRNAs belong to a class of small regulatory single-stranded RNA molecules, ranging from 21 to 23 nucleotides in length, the roles of which in development and disease are increasingly acknowledged. They act as gene expression down-regulators, altering the stability or translational efficiency of messenger RNAs (mRNAs) with which they share partial sequence complementarity, and are predicted to affect up to one- third of all human genes. miRNA molecules are encoded by genes, from the DNA of which they are transcribed but not translated into protein
  • non-coding RNA each primary transcript (a pri-miRNA) is processed into a short stem-loop structure called a pre- miRNA and finally into a mature and functional miRNA.
  • miRNA analyses by means of microarray techniques have the drawback resulting from the different melting temperatures (hereinafter referred to as Tms) and therefore the problem of the thermal stability of the hybrids which are formed. This situation is the result of the high variability of the CG content of the miRNAs . As may be apparent, this problem may compromise the overall analysis technique, as it does not provide the required selectivity criteria .
  • Figure 1 and Figure 2 show the operation sequences of the microarray which is the object of the present invention respectively with a target miRNA and a miRNA other than the target miRNA;
  • Figure 3 shows the replicating step for generating the probe strand
  • Figure 4 shows the probe strand bound to the substrate for generating the microarray which is the object of the present invention.
  • Figure 5 shows the secondary structure of the hairpin structure of a probe strand of the microarray which is the object of the present invention.
  • numeral 1 indicates a probe hybrid bound to a substrate 2.
  • the probe hybrid consists of a labelling segment 3 which has a fluorophore moiety 4 bound thereto, and of a probe strand 5 bound to substrate 2.
  • Probe strand 5 consists of a hairpin structure 6, a probe sequence 7 adapted to selectively bind the miRNA to be analysed and occurring as a single strand, and a complementary labelling sequence 8 hybridised in use to labelling segment 3.
  • probe hybrid 1 is bound to substrate 2 to generate the microarray which is the object of the present invention.
  • probe hybrid 1 specifically binds target miRNA 9 by means of probe sequence 7.
  • a ligating step is performed in order to ligate target miRNA 9 to labelling segment 3 and to the 5' phosphate end of hairpin structure 6.
  • a washing step is performed to dehybridise labelling segment 3 from complementary labelling sequence 8.
  • the ligating step successfully ligates labelling segment 3 to target miRNA 9.
  • Labelling segment 3 thereby remains bound to probe strand 5 even following the step of washing and may therefore be detected by means of the common fluorescence techniques in virtue of the presence of fluorophore moiety 4.
  • the miRNA bound to probe sequence 7 is not target miRNA 9 but instead a different miRNA 9a, the latter will have regions X which are not complementary to probe sequence 7.
  • the presence of these non-complementary regions X impairs the action of the ligating reaction and therefore ligation of labelling segment 3 and miRNA 9a.
  • the absence of this ligation results in labelling segment 3 not remaining bound to probe strand 7 following the step of washing and therefore not being detected as fluorophore moiety 4 is missing.
  • a variant (not shown in the Figures) of the probe strand of the present invention relates to a strand that does not have hairpin structure 6, but instead comprises a complementary ligating sequence hybridised in use to a ligating segment. In other terms, the hybridisation between the complementary ligating sequence and the ligating segment replaces hairpin structure 6.
  • FIG. 3 shows a template strand 10 which is formed by a first sequence 11a complementary to a promoter sequence disclosed hereafter and attached to a substrate 12, a hairpin sequence lib, a sequence lie identical to target miRNA 9 and a sequence Hd identical to labelling sequence 3.
  • a replication is performed on template strand 10 ( Figures 3A and 3B) from a promoter sequence 13, which has a free 3' -phosphate end and comprises a portion 14 complementary to hairpin sequence Hb to subsequently generate hairpin structure 6.
  • Portion 14 complementary to hairpin sequence Hb ends with a free 5' phosphate end and is bound to a functional moiety 15 to allow stamping according to the SuNS technique.
  • promoter sequence 13 hybridises by its
  • Figure 4 and forming the microarray object of the present invention together with labelling segments 3, are generated in this manner.
  • the promoter strand does not comprise portion 14, whereas the sequence disclosed as hairpin sequence 6 is a sequence identical to that of the ligating segment that will hybridise to the strand generated by replication . Furthermore, stamping is not necessarily performed according to the SuNS technique.
  • Hairpin of length between 20-40 nucleotides allowing to create a rigid probe structure on the microarray surface; Tm between 75-85°C and GC content between 70-90%: the described structure must be stable over a wide range of temperatures (20-90 0 C), in order to avoid any, even if partial, denaturation, allowing for a specific sample hybridization; the hairpin structure must be the only possible structure for the described nucleotide sequence .
  • - Detector sequence of length between 15-20 nucleotides. It was designed with a Tm of about 45-55°C and 50-60% GC content. Such characteristics were found to allow for better simultaneous hybridization of both miRNA sample and detector, without being limited by their respective Tms .
  • - SuNS ARRAY PREPARATION - Generated probe 5 has a hairpin sequence 6 (SEQ ID NO:1) having a Tm of 78.9°C and a GC content of 81.8% and a complementary detector sequence 8 (SEQ ID NO: 2) having a Tm of 48.4°C and a GC content of 57.1%, to be hybridised to a universal detector sequence (SEQ ID NO: 3) .
  • a specific miRNA binding sequence 7 is located between hairpin sequence 6 and complementary detector sequence 8.
  • SuNS template 10 used to generate the above mentioned probe 5 has a primer binding sequence 11a (SEQ ID NO: 4) having a Tm of 49.3°C and a GC content of 60%, a hairpin sequence lib (SEQ ID NO: 5) and a detector sequence Hd (SEQ ID NO: 3) .
  • a specific miRNA sequence lie is located between hairpin sequence lib and detector sequence Hd.
  • the hairpin secondary structure at 37°C is shown in Figure 5.
  • SEQ ID NO:1 GCCCGGGCGCCGTTCAAGAGACGGCGCCCGGGC SEQ ID NO: 2: CATGTGTCGTGCCT SEQ ID NO: 3: AGGCACGACACATG SEQ ID NO: 4: CCGTCTCTTGAACGG SEQ ID NO: 5: GCCCGGGCG - miRNA DETECTION -
  • the experimental procedure used for miRNA detection is as follows.
  • Sample Hybridization procedure An equimolar aliquot of purified and 5' phosporilated miRNA (or total RNA) sample and universal labeled-detector were prepared in IX hybridization buffer (final concentration: 1 ⁇ M) .
  • the sample was denatured at 90 0 C for 2 min and hybridization was carried on a Tecan HS Pro hybridization station, using the following protocol:
  • T4-ligase 0.3 U/ ⁇ l
  • T4-ligase buffer IX Final volume: 150 ⁇ l .
  • the ligase-reaction was carried out on a Tecan hybridization station, using the following protocol: - Slide wash at 30°C, with SSC 0. IX, 20 sec soak + 20 sec flow;
  • the described protocol includes the stringent wash at 70 0 C that allows for labeled-detector removal in case of a mismatch introduced by an incorrect miRNA hybridization.
  • the analysis technique for miRNAs with microarrays which is the object of the present invention, does not provide for the labelling of the target miRNA, which is usually the most critical step during RNA manipulation, and does not depend on the Tm of the miRNA hybrids, as dehybridisation concerns the hybrid formed by labelling segment 3. Furthermore, such a method of detection has the advantages relating to the use of total RNA without first having to purify miRNAs, and to the fact that the ligation reaction introduces a level of specificity that cannot be achieved by hybridization alone.
  • the microarray object of the present invention may be applied not only to the analysis of miRNA but also to any nucleotide sequence for the most various purposes, for instance also for genotyping DNA.

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

Abstract

L'invention porte sur un microréseau pour l'analyse de séquences nucléotidiques simple brin, en particulier de miARN comprenant une pluralité de brins sondes (5) fixés à un substrat (2) et aptes à former des hybrides de sonde respectifs (1) avec un segment de marquage (3) lié à une fraction fluorophore (4) et avec un segment de ligature. Chacun des brins sondes (5) est constitué d'une séquence de ligature complémentaire hybridée à l'utilisation au segment de ligature d'une séquence de marquage complémentaire (8) hybridée à l'utilisation au segment de marquage (3) et d'une séquence de sonde (7) placée entre la séquence de ligature complémentaire et la séquence de marquage complémentaire (8) et apte à ligaturer sélectivement une séquence nucléotidique simple brin cible.
EP09709868A 2008-02-15 2009-02-13 Microréseau pour l'analyse de séquences nucléotidiques Withdrawn EP2250284A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
IT000103A ITBO20080103A1 (it) 2008-02-15 2008-02-15 Microarray per l'analisi di sequenze nucleotidiche
PCT/EP2009/051738 WO2009101193A1 (fr) 2008-02-15 2009-02-13 Microréseau pour l'analyse de séquences nucléotidiques

Publications (1)

Publication Number Publication Date
EP2250284A1 true EP2250284A1 (fr) 2010-11-17

Family

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Family Applications (1)

Application Number Title Priority Date Filing Date
EP09709868A Withdrawn EP2250284A1 (fr) 2008-02-15 2009-02-13 Microréseau pour l'analyse de séquences nucléotidiques

Country Status (4)

Country Link
EP (1) EP2250284A1 (fr)
CA (1) CA2715538A1 (fr)
IT (1) ITBO20080103A1 (fr)
WO (1) WO2009101193A1 (fr)

Families Citing this family (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2785529C (fr) * 2010-02-11 2019-01-08 Nanostring Technologies, Inc. Compositions et procedes de detection de petits arn
US20110257385A1 (en) * 2010-02-23 2011-10-20 Life Technologies Corporation Methods for flip-strand immobilizing and sequencing nucleic acids
WO2011157617A1 (fr) * 2010-06-17 2011-12-22 Febit Holding Gmbh Ensemble complexe de banques de miarn
DE102012204366B4 (de) * 2011-12-16 2017-07-27 Siemens Aktiengesellschaft Verfahren und Kit zur Identifizierung und Quantifizierung von einer einzelsträngigen Ziel-Nukleinsäure
EP2837695B1 (fr) * 2012-04-12 2018-01-10 The University of Tokyo Procédé de quantification d'acide nucléique, sonde de détection, jeu de sondes de détection, et procédé de détection d'acide nucléique
DE102013221402A1 (de) * 2013-10-22 2015-04-23 Siemens Aktiengesellschaft Verfahren zur Detektion und Quantifizierung von einer einzelsträngigen Ziel-Nukleinsäure
WO2015069787A1 (fr) * 2013-11-05 2015-05-14 Htg Molecular Diagnostics, Inc. Procédés de détection d'acides nucléiques
WO2016010047A1 (fr) * 2014-07-18 2016-01-21 国立大学法人東京大学 Procédé de détection d'un acide nucléique, sonde de capture, jeu de sondes de détection, micro-réseau, kit de détection d'un acide nucléique, acide nucléique immobilisé sur phase solide, et dispositif fluidique

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007117256A1 (fr) * 2005-05-31 2007-10-18 Applera Corporation Amplification multiplexée d'acides nucléiques courts
EP1924713B1 (fr) * 2005-08-24 2011-11-09 Life Technologies Corporation Procédé permettant de quantifier des arnsi, arnmi et des arnmi polymorphes
US20070172841A1 (en) * 2006-01-25 2007-07-26 Hui Wang Probe/target stabilization with add-in oligo
US20080038727A1 (en) * 2006-03-10 2008-02-14 Applera Corporation MicroRNA and Messenger RNA Detection on Arrays

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO2009101193A1 *

Also Published As

Publication number Publication date
WO2009101193A1 (fr) 2009-08-20
CA2715538A1 (fr) 2009-08-20
ITBO20080103A1 (it) 2009-08-16

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