WO2000014273A2 - Technique de representation de differentiels genetiques et vecteur - Google Patents

Technique de representation de differentiels genetiques et vecteur Download PDF

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Publication number
WO2000014273A2
WO2000014273A2 PCT/CA1999/000789 CA9900789W WO0014273A2 WO 2000014273 A2 WO2000014273 A2 WO 2000014273A2 CA 9900789 W CA9900789 W CA 9900789W WO 0014273 A2 WO0014273 A2 WO 0014273A2
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Prior art keywords
length
mono
library
gene
complementary
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PCT/CA1999/000789
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English (en)
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WO2000014273A3 (fr
Inventor
Abdel Majid Belouchi
Hélène FOURNIER
Mark Gee
Denis Gauvreau
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Signalgene Inc.
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Priority to CA002342903A priority Critical patent/CA2342903A1/fr
Publication of WO2000014273A2 publication Critical patent/WO2000014273A2/fr
Publication of WO2000014273A3 publication Critical patent/WO2000014273A3/fr

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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
    • C12Q1/6809—Methods for determination or identification of nucleic acids involving differential detection

Definitions

  • This invention discloses a method of determining differential display
  • RNA expression profiles in multiple samples by the creation of an RNA fingerprint for each sample by the creation of an RNA fingerprint for each sample. Briefly, partial complementary cDNA
  • sequences are amplified from subsets of mRNAs by reverse transcription
  • cDNA is used as a template in PCRs
  • Radiolabeled nucleotide is incorporated into the display
  • Regions corresponding to bands of interest are excised from the dried gel
  • Clones can then be used as probes to confirm differential
  • microarrays are used to analyze the expression of large numbers of genes in
  • oligonucleotide probe For example, oligonucleotide probe
  • This invention comprises a method of determining differential display
  • substantially partial-length segment library corresponding to or
  • step (f) using the library of step (e) and preparing like-condition mono-
  • length second cRNA library being a substantially mono-length segment
  • sequences of the second gene source wherein said library is a substantially
  • an expression-length library is a corresponding sequence.
  • differential is noted to be a nucleotide binding differential.
  • remote-site restriction enzyme such as with Bpm I. It is understood that
  • Such libraries can be either cRNA or cDNA depending on the procedures
  • gene sources for expressed cellular cDNA are provided.
  • RNA from mRNA are from cells in either affected or non-affected states. Particular note is made of mono-length sequences that are cRNA
  • the provided nucleotide portion comprises ⁇ '-gcuggagaucgg-
  • the inquiry portion comprises
  • a constant and a variable portion and the variable portion is at least about
  • inquiry nucleotide portion is about 3-mer. In some such embodiments it is
  • the provided portion comprises 5'cuggag3' . It is further
  • the inquiry portion has a 3'-end and a 5'-end, and said
  • 3'-end is 5'cgg3'.
  • the inquiry portion is useful in a
  • variable portion in a range of from about 1 - to 20-mer, the variable portion in a range of from
  • nucleotide segments of the target array as usefully from
  • the method also includes the differential being determined by
  • comparison reporters selected from the group consisting of
  • a substrate accessibly affixed to a substrate, optionally on a nylon membrane, or a
  • Oligonucleotides are usefully attached to substrate by way of an
  • intervening spacer molecule of at least about 6-carbons, or at least about a
  • the invention comprises a substantially
  • mono-length segments are about 14-mer with about 1 1 -mer corresponding
  • the invention includes a vector-insertable
  • linker (such as a BssH II recognition site adjacent to) comprising a promoter
  • vector-insertable linker is pBluescript II SK + cloning vector.
  • T7 promoter site which is adjacent to a Bss Hll recognition site.
  • the invention comprises a mono-length
  • n represents one of 1 1 nucleotides in sequence corresponding to a
  • the invention includes a method of
  • determining differential display of gene expression comprising the steps of:
  • step (c) using the library of step (b) and preparing mono-length first cDNA
  • substantially partial-length segment library corresponding to or
  • step (e) using the library of step (e) and preparing like-condition mono-
  • length second cRNA library being a substantially mono-length segment
  • step (a) each with an ordered array of step (a); and (g) comparing the probe hybridized accessible ordered arrays to
  • nucleotide sequence data base to identify the gene of expression
  • the invention includes a method of detecting a point mismatch probe
  • segment or expression-length segment nucleotide libraries comprising
  • probe nucleotides and the target nucleotides are of
  • the probe nucleotide has a constant portion and an inquiry portion
  • target site oligonucleotide has a portion complementary to the constant
  • gene tag library wherein the mono-length gene tags are either cDNA or
  • the mono- length insert segments are in specific embodiments
  • 1 -mer or 14-bp segments respectively correspond to or are
  • mono-length segments are
  • FIG. 1 is a diagrammatic flow chart of the differential display
  • Fig. 2(A)-(D) provides sequence information for specific vector
  • Fig. 3. is a flow chart of procedures in a particular embodiment.
  • Fig. 4(A), (B) and (C) depict accessible array test results.
  • A. Gene is a unit of genetic information. In one embodiment it is a region
  • genes can reside in
  • non-coding sequences (known as exons and introns, respectively)
  • promoters e.g., promoters, enhancers, operons, and other regulatory regions.
  • viruses including without limitation, viruses, bacteria, fungi, plants, and
  • animals including humans, and further including neoplasms and cell
  • RNA expressed in a living cell or in a cellular system shall mean the mRNA expressed in a living cell or in a cellular system
  • the mRNA expressed is then translated into complementary DNA (cDNA) for differential diagnostic
  • DD is capable of monitoring a wide range of expressed
  • DD represents a functional genomics approach
  • Deregulation may involve gross or subtle changes in gene expression
  • under-expression including zero expression
  • over-expression including zero expression
  • Affected cells shall mean cells which are diseased, or
  • gene induction or repressor agents e.g. agents which through
  • chemical toxins e.g. chemical compounds which through their
  • disease or resistance state e.g. susceptibility to disease or
  • tissue specificity e.g. expression which is specific to brain
  • strain or varietal specificity e.g. particularly in Ag-bio
  • rary shall mean substantially the totality of genetic material in the
  • an expressed gene library shall represent substantially the totality of the expressed genes. It is
  • stantially mono-length segment library shall mean a library
  • gene tag probes either cDNA or cRNA, that are short -
  • Such mono-length segments function as
  • Mono-lengths of cRNA are used for
  • nucleotide portion shall mean the nucleotide sequence specific
  • 5'g3' is a provided nucleotide portion; the first base to be
  • T3 RNA polymerase promoter site is
  • nucleotides on the opposite 3' strand are nucleotides on the opposite 3' strand.
  • recognition site is 5'atcgat.
  • 5'cgg3' is the constant moiety of the inquiry nucleotide portion.
  • the inquiry portion is then a
  • n a (adenine), g
  • nucleotide H. Inquiry nucleotide portion shall mean the nucleotide sequence specific
  • nucleotide portion is variable for any given probe generated from a
  • short mono-length segments, that is from about 1 1 -
  • segments consist of a "provided nucleotide portion" of from about
  • a provided nucleotide portion is made
  • the inquiry portion can be any suitable item.
  • variable moiety is noted in particular
  • Partial-length segment library shall mean a cDNA library generated by
  • RNAs By way of example, Msp I - Not I DNA
  • fragments cloned into the pALLgenes vector constitute a partial-length segment library.
  • the partial-length segment library is,
  • Msp I shall mean the restriction endonuclease or enzyme
  • Cla I is the restriction endonuclease or
  • T3 initiates synthesis of RNA in a particular
  • RNA polymerases include bacteriophage SP6 and T7 DNA-dependent
  • ibrary of substantially expression length sequences shall mean a
  • transcribed to cDNA is conveniently cloned into a suitable phage
  • RNA sequence using a back figuring strategy (Fig. 3). Using this
  • library contains substantially full-length cDNA sequencing templates.
  • RNA is relatively less stable than DNA and may possess
  • templates to be substantially, as opposed to completely, full-length.
  • K. DNA shall be expansively understood to mean a molecule of
  • deoxyribonucleic acid comprising the component purines and
  • genomic sources e.g., from the cell nuclei of living
  • recombinant vectors e.g., plasmids, phage, cosmids,
  • polymerized sequences such as PCR products, sequencing reaction
  • a string of DNA is polymerized by addition of
  • nucleotides are broadly branched branched branched branched branched branched branched branched branched branched branched branched branched branched branched branched branched branched branched branched branched branched branched branched branched branched branched branched branched branched branched branched branched branched branched nucleotides. In some instances, the term nucleotides is broadly branched branched nucleotides.
  • RNA shall be expansively understood to mean a molecule of ribonucleic
  • RNA is generally transcribed from DNA. mRNA is single-
  • stranded and normally contains a poly-A tail i.e., a string of
  • RNA RNA is polymerized by addition
  • replication e.g. induced vs. non-induced, diseased vs. normal, trait-
  • N. bp and -mer shall mean the abbreviated forms of the terms base pair(s)
  • oligomers and oligomer(s), respectively.
  • the number of oligomers shall mean
  • RNA string refers to a single-stranded
  • a base pair refers to
  • a 20-bp fragment refers to a
  • double-stranded DNA sequence comprising 20 complementary bases.
  • Remote-site restriction endonucleases shall mean enzymes which do
  • Remote-site restriction enzymes typically do
  • An ordered array or oligonucleotide array detector refers to an ordered
  • oligonucleotides means a collection of oligonucleotides which comprise
  • Substrate shall mean any solid phase surface to which
  • oligonucleotides whether with or without linker or spacer groups, may be
  • Attachment methods e.g., nylon membrane, glass slide, biochip. Attachment methods
  • Accessible array shall mean that the oligonucleotides of an ordered
  • a carbon spacer molecule refers
  • Indicator of complementarity shall mean a reporter system for the
  • the reporter may be a signal emittor
  • a radioactive label such as a radioactive label, fluorescent label or chemiluminescent label physically attached to the probe so as to permit signal detection
  • photographic paper or other signal detection system In some embodiments, photographic paper or other signal detection system.
  • the indicator of complementarity is of sufficient
  • S. Incremental wash conditions shall mean washing the hybridized array
  • T. Target shall mean a DNA sequence immobilized onto a solid phase
  • the target shall be a
  • the target is not always of identical length
  • target hybridization is augmented with "blocker" oligos added during
  • Gene site anomaly shall mean a DNA sequence which is deviant from
  • SNP polymorphisms
  • a gene site anomaly In some instances, a gene site anomaly
  • Fig 3 discloses one line of cells to be compared as to affected and non-
  • mRNA is extracted and reverse-
  • This cDNA is then used to generate an expression-length
  • RNA polymerase to generate a short mono-length library consisting of
  • cRNA probes are used to hybridize against a set of
  • the technology entails generating a library of
  • oligonucleotides are conveniently immobilized onto a substrate
  • substrate such as membrane, a chip, or the like to provide stable loci for an
  • analysis is performed as described in U.S.
  • primer sequences respectively corresponding to a) the gene tag sequence of
  • the invention comprises generating a
  • oligonucleotides immobilized onto a membrane or a chip immobilized onto a membrane or a chip. The presence or
  • RNA sequence is subsequently retrieved from
  • a specialized cloning vector, pALLgenesTM is
  • This specialized vector is a modified pBluescript II SK
  • cloning vectors with replacable linkers are also useful, including by way of
  • pGEM pUC1 9, and pBR322.
  • recognition sites arranged in the proper spatial arrangement, so as to
  • probe length is shortened if the choice of enzymes is one in which their
  • the enzymes are selectively engineered such that they will be
  • the pALLgenes vector is useful in generating a library of short mono-length
  • variable length probe Having a tightly restricted range of probe and target length reduces the overall complexity and randomness of
  • this technology in one embodiment represent the 3' end of expressed gene
  • the Bpm I enzyme can also be used to
  • nucleotide sequence of the oligo target is exposed and accessible to the
  • hybridization is further increased by subjecting the hybridized membrane or
  • target hybridization is very high and is able to discriminate down to single
  • every 0.5°C to about 1 °C, or about 3 °C are useful for single base
  • the present invention permits
  • hybridization-based differential display techniques that are, for example,
  • complex organisms such as humans, contain between 20,000 to 30,000
  • restriction enzymes can be used at particular stages of
  • the invention is not restricted to Not I and Msp I and
  • Not I enzyme is a rare-cutter.
  • Msp I An important property of Msp I is that it is a frequent-cutter and has a
  • CpG islands are associated with regions of the genome that are
  • Cla I is replaced by one for either BamH I or EcoR V.
  • inserts are generated by digestion with 1 ) Sau3A I or 2) Alu I,
  • the present invention also detects the DNA
  • hybridizing gene tags is known (i.e. location of signal on the ordered array),
  • the PCR product is
  • a suitable primer is designed using the
  • a suitable primer is an oligonucleotide
  • the template DNA including having minimal secondary structure
  • Such primers are about 1 8- to 23-mer in length and
  • the phage vector are used to selectively amplify by PCR the substantially
  • the signal detection system e.g. Phospholmager from Molecular
  • oligonucleotide sequences are
  • Attachment of the oligonucleotide is usefully carried out in a such a way so
  • target nucleotide sequence to float above the membrane surface ands thus
  • the substrate is more useful in a format that is
  • An array serves as a fixed matrix of possible permutations of
  • tissue specificity tissue specificity, developmental specificity, or mutation. It is particularly desirable
  • a significant use of the present invention is in gene identification
  • expression profiling and monitoring provides a non-ambiguous portrait of multiple genes expressed simultaneously in response to a given stimulus
  • the target array configuration remains
  • the present invention is usefully employed in drug and toxicology
  • Oligos B, C and D contained 1 , 2 and 3
  • Oligo F contained a
  • Oligo G was at-rich. Oligo H was gc-rich.
  • the probe oligos were radioactively end-labeled with 3 P,
  • Oligos with C- spacers had a primary reactive amine group added to the 5' ends via a
  • Type #1 a negatively charged membrane
  • Radioactive probes (7.5 x 1 0 5 cpm/mL) were denatured by
  • Wells 3, 1 2, 1 5, 1 8, 21 , 24 contained the specific oligo of
  • Probes consisted of complementary oligos with no
  • the objective was to construct a cloning vector with the promoter
  • the vector was a modified pBluescript II SK + (Short, J.M., et al.,
  • polylinker containing, in order, the following restriction or polymerase
  • promoter recognition sites Bss Hll - T3 promoter site - Bpm I - Cla I - Not I
  • oligos were designed to anneal together at a short internal complementary
  • Oligos I and J were used as a template in a PCR
  • Oligo I 500 ng Oligo J, 1 00 uM dNTP, 1 .2 mM MgCI2, 2 U Taq (Gibco,

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

Abstract

L'invention porte sur un procédé permettant d'obtenir une représentation différentielle de l'expression de gènes par comparaison de bibliothèques d'ARNc mono-longueurs. Ces bibliothèques sont hybridées par des sondes en réseaux ordonnés accessibles permettant de reconnaître les sites présentant des hybridations parmi les bibliothèques de segments mono-longueurs, et de localiser les gènes présentant un différentiel d'expression. L'invention porte également sur une bibliothèque de mono-longueurs correspondant à des, ou complémentaires de, séquences d'ARNm exprimé, comportant les éléments d'ARNc de 23 nucléotides de long et de formule 5'-gcuggagaucggnnnnnnnnnnn-3', dans laquelle n représente l'un des 11 ribonucléotides de la séquence correspondant à la séquence complémentaire de 11 nucléotides de l'ARNm.
PCT/CA1999/000789 1998-09-03 1999-08-26 Technique de representation de differentiels genetiques et vecteur WO2000014273A2 (fr)

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CA002342903A CA2342903A1 (fr) 1998-09-03 1999-08-26 Technique de representation de differentiels genetiques et vecteur

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001081625A1 (fr) * 2000-04-26 2001-11-01 Ipsogen Methode de mesure quantitative de l'expression des genes
WO2005001135A2 (fr) * 2003-06-03 2005-01-06 Arcturus Bioscience, Inc. Detection d'acides nucleiques induite en 3'

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997013877A1 (fr) * 1995-10-12 1997-04-17 Lynx Therapeutics, Inc. Mesure de profils d'expression genique pour evaluer la toxicite
US5658736A (en) * 1996-01-16 1997-08-19 Genetics Institute, Inc. Oligonucleotide population preparation
WO1998015651A1 (fr) * 1996-10-04 1998-04-16 Brax Genomics Limited Identification de la liaison d'un oligonucleotide non codant

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997013877A1 (fr) * 1995-10-12 1997-04-17 Lynx Therapeutics, Inc. Mesure de profils d'expression genique pour evaluer la toxicite
US5658736A (en) * 1996-01-16 1997-08-19 Genetics Institute, Inc. Oligonucleotide population preparation
WO1998015651A1 (fr) * 1996-10-04 1998-04-16 Brax Genomics Limited Identification de la liaison d'un oligonucleotide non codant

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
LIANG P ET AL: "DIFFERENTIAL DISPLAY OF EUKARYOTIC MESSENGER RNA BY MEANS OF THE POLYMERASE CHAIN REACTION" SCIENCE,US,AMERICAN ASSOCIATION FOR THE ADVANCEMENT OF SCIENCE,, vol. 257, no. 5072, 14 August 1992 (1992-08-14), pages 967-971, XP000508268 ISSN: 0036-8075 cited in the application *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001081625A1 (fr) * 2000-04-26 2001-11-01 Ipsogen Methode de mesure quantitative de l'expression des genes
FR2808287A1 (fr) * 2000-04-26 2001-11-02 Ipsogen Methode de mesure quantitative de l'expression des genes
WO2005001135A2 (fr) * 2003-06-03 2005-01-06 Arcturus Bioscience, Inc. Detection d'acides nucleiques induite en 3'
WO2005001135A3 (fr) * 2003-06-03 2005-02-03 Arcturus Bioscience Inc Detection d'acides nucleiques induite en 3'

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Publication number Publication date
CA2342903A1 (fr) 2000-03-16
WO2000014273A3 (fr) 2000-06-02

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