EP1121470A1 - Procedes de manipulation de populations d'acides nucleiques au moyen d'oligonucleotides a marquage peptidique - Google Patents

Procedes de manipulation de populations d'acides nucleiques au moyen d'oligonucleotides a marquage peptidique

Info

Publication number
EP1121470A1
EP1121470A1 EP99954899A EP99954899A EP1121470A1 EP 1121470 A1 EP1121470 A1 EP 1121470A1 EP 99954899 A EP99954899 A EP 99954899A EP 99954899 A EP99954899 A EP 99954899A EP 1121470 A1 EP1121470 A1 EP 1121470A1
Authority
EP
European Patent Office
Prior art keywords
label
cdna
library
dna
peptide
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Withdrawn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
EP99954899A
Other languages
German (de)
English (en)
Inventor
Francois J.-M. Iris
Jean-Louis Pourny
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.)
Valigen US Inc
Original Assignee
ValiGene Corp
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 ValiGene Corp filed Critical ValiGene Corp
Publication of EP1121470A1 publication Critical patent/EP1121470A1/fr
Withdrawn legal-status Critical Current

Links

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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1072—Differential gene expression library synthesis, e.g. subtracted libraries, differential screening
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1065—Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags
    • 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

  • 15 major advantage of the methods of this invention is the ability to screen multiple populations of cDNAs derived, for example, from different tissues belonging to the same individual or to phenotypically different cell types present concurrently within a given tissue sample.
  • This invention further provides in the fourth embodiment additional methods wherein the DNA hybridized from each of the plurality of other cDNA libraries is in excess relative to the first cDNA library. Furthermore, additional methods are provided which c employ from a 2-fold to a 100-fold excess, from a 2.5-fold to a 10-fold excess and wherein the excess is a 3-fold excess.
  • RNA:DNA hybrid produced by cDNA first-strand synthesis, including the target-specific primer on the 5' end of the cDNA strand.
  • the quantity of each PCR reaction product used in the hybidization reaction can vary. Since isolation of cDNA fragments specific to one library is desired, the other libraries are used in excess (i.e., as a "mop"). Specifically, where one wishes to isolate fragments present in library A but not in B, C, D and E, one uses excess B, C, D and
  • the material trapped in the first column will contain all fragments tagged with B, C, D and E labels. This will include hybrids containing one A-tagged strand. This 0 material can also be eluted and further sorted using other embodiments of the invention, or as otherwise desired by the practitioner.
  • the inserts from each of a plurality of cDNA libraries are linearized and tagged by PCR with a distinguishable library-specific label attached to vector-specific primers. Accordingly, as above, all fragments produced have identical nucleotide sequences at their 5' and 3' ends corresponding to the vector-specific primer sequences, and a distinguishable label indicating the library-of-origin.
  • the labeled PCR products are then, purified by exclusion chromatography to remove all reaction components, including excess labeled primers.
  • the purified, labeled PCR products are then combined, heat-denatured and allowed to reanneal.
  • each Phase II column captures single strands bearing one of the distinguishable peptide labels.
  • Each Phase II column is then separately eluted. In this way, an array of twenty groups of single-stranded cDNA fragments is isolated wherein each of the twenty groups contains fragments shared (i.e. hybridizable) between two libraries (see FIG. 1, Phase II).
  • All the independent members belonging to a given library Group are 5 likewise amplified and labeled by PCR.
  • the amplified products are then mixed, denatured and allowed to re-anneal.
  • the reannealed Phase III input mixture is then divided into four aliquots. The number of aliquots needed depends on the number of separate labels in the mixture.
  • the A library Group is analyzed and the labels used during the PCR Q amplifying process are B, C, D and E.
  • the Phase III columns consist of four series of affinity columns, each series consisting of three single-antibody columns. Each of these four series of columns contain antibodies specific to three of the four labels used in the Phase III PCR amplification step.
  • the four series of affinity columns contain: 5 Series 1 - C, D and E antibodies;
  • both single-stranded fragments and double- stranded library plasmids share identical extremities (i.e., 5' and 3' ends) over at least 10-15 bases, and the homologous fragments are at least 350 bp in length. If strong overall 5 homology is present, perfect identity between fragments is not required for RecA to form stable triple-stranded structures (see, e.g., Rao et al.,1995, Trends In Biological Science 20: 109-113).
  • the cloned inserts do not exceed 1-2 kilobase (kb) in length so that clones sharing only strong localized homologies with the subtraction probes are not selected.
  • Mung bean nuclease in the amount of 1.0 unit per microgram of DNA:RNA hybrid is added and the mixture is incubated at 30 °C for thirty (30) minutes.
  • the enzymes 5 may then be inactivated by phenol/chloroform extraction or by addition of SDS to 0.01%.
  • the blunt-ended hybrids may be recovered by alcohol precipitation.
  • the sample is now purified by standard exclusion chromatography. After purification, the sample consists of the DNA:RNA hybrids together with the remainder of o the total RNA species initially present. The exclusion chromatography removes the small
  • the amount of product produced by the PCR reaction can now be quantified 5 by a modification of an enzyme-linked immunoassay technique (ELISA).
  • the purified reaction mixture may be analyzed in microtiter wells coated with an antibody specific to the peptide label that was attached to the target-specific primer. Streptavidin-linked horseradish peroxidase can then be added to bind to the biotin moiety attached to the standard primer of the retained PCR products. A horseradish peroxidase substrate can then be added, and the reaction product quantified (see e.g. Sambrook et al, 1989, Molecular Cloning A Laboratory Manual. 2nd Ed., Cold Spring Harbor Laboratory Press at 18.75), indicating the amount of target mRNA present in the original sample.
  • ELISA enzyme-linked immunoassay technique
  • Spacer Phosphoramidite C3 i.e., 3-O-Dimethoxytrityl-propyl-l -[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite
  • Spacer Phosphoramidite C3 3-O-Dimethoxytrityl-propyl-l -[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite
  • Ohgonucleotides may be labeled with a wide variety of lables for use in the various embodiments of the invention.
  • European Patent Publication No. EP 5 0370 694 A2 entitled, “Diagnostic Kit and Method Using a Solid Phase Capture Means For Detecting Nucleic Acid", by Burdick and Oakes, publication date May 30, 1990, discloses methods of linking labels to ohgonucleotides.

Landscapes

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

Abstract

L'invention concerne généralement des procédés de marquage, de tri et de criblage de populations d'acides nucléiques. Elle porte notamment sur un procédé de tri et de comparaison de populations complexes d'acides nucléiques, telle que des banques d'ADNc. Ces populations complexes peuvent être dérivées de cellules ou de types de tissus comportant des variations du phénotype présentant un éventuel intérêt clinique. Le procédé est généralement appelé VG-PLOSM, procédé à Oligonucléotides à marquage peptidique, ou et implique l'utilisation d'étiquettes peptidiques identifiables, liées à des amorces olignonucléotidiques identiques.
EP99954899A 1998-10-16 1999-10-15 Procedes de manipulation de populations d'acides nucleiques au moyen d'oligonucleotides a marquage peptidique Withdrawn EP1121470A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US17432898A 1998-10-16 1998-10-16
US174328 1998-10-16
PCT/US1999/023906 WO2000023622A1 (fr) 1998-10-16 1999-10-15 Procedes de manipulation de populations d'acides nucleiques au moyen d'oligonucleotides a marquage peptidique

Publications (1)

Publication Number Publication Date
EP1121470A1 true EP1121470A1 (fr) 2001-08-08

Family

ID=22635773

Family Applications (1)

Application Number Title Priority Date Filing Date
EP99954899A Withdrawn EP1121470A1 (fr) 1998-10-16 1999-10-15 Procedes de manipulation de populations d'acides nucleiques au moyen d'oligonucleotides a marquage peptidique

Country Status (7)

Country Link
EP (1) EP1121470A1 (fr)
JP (1) JP2002527118A (fr)
KR (1) KR20010102909A (fr)
CN (1) CN1342208A (fr)
AU (1) AU1113000A (fr)
CA (1) CA2344625A1 (fr)
WO (1) WO2000023622A1 (fr)

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2419159A1 (fr) * 2000-08-11 2002-02-21 Agilix Corporation Systemes de detection ultrasensibles
KR101110013B1 (ko) * 2007-10-05 2012-02-29 (주)바이오니아 서열 내에 어베이직 부분을 포함하는 ｐｃｒ 증폭용프라이머
US20120308570A1 (en) * 2009-05-08 2012-12-06 Tatiana Kolesnik Methods for treating diseases
CA3233475A1 (fr) * 2021-09-30 2023-04-06 Claire Bevis-Mott Methodes de blocage

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1989001526A1 (fr) * 1987-08-07 1989-02-23 Genelabs Incorporated Librairie et procede de clonage par coincidence
US5804382A (en) * 1996-05-10 1998-09-08 Beth Israel Deaconess Medical Center, Inc. Methods for identifying differentially expressed genes and differences between genomic nucleic acid sequences
JPH11509427A (ja) * 1996-05-14 1999-08-24 ベーリンガー マンハイム ゲーエムベーハー 試料中の核酸分子の発現の同定方法および／または定量方法
US6221585B1 (en) * 1998-01-15 2001-04-24 Valigen, Inc. Method for identifying genes underlying defined phenotypes

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO0023622A1 *

Also Published As

Publication number Publication date
WO2000023622A1 (fr) 2000-04-27
JP2002527118A (ja) 2002-08-27
KR20010102909A (ko) 2001-11-17
CN1342208A (zh) 2002-03-27
CA2344625A1 (fr) 2000-04-27
AU1113000A (en) 2000-05-08

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