EP1117826A1 - Amorces oligonucleotidiques destabilisant la formation de duplex non specifiques et leurs utilisations - Google Patents

Amorces oligonucleotidiques destabilisant la formation de duplex non specifiques et leurs utilisations

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Publication number
EP1117826A1
EP1117826A1 EP99947148A EP99947148A EP1117826A1 EP 1117826 A1 EP1117826 A1 EP 1117826A1 EP 99947148 A EP99947148 A EP 99947148A EP 99947148 A EP99947148 A EP 99947148A EP 1117826 A1 EP1117826 A1 EP 1117826A1
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Prior art keywords
modification
sequence
homopolymeric
modified oligonucleotide
modified
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German (de)
English (en)
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Jerry Pelletier
Manjula Das
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McGill University
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McGill University
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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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6853—Nucleic acid amplification reactions using modified primers or templates
    • 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
    • C12Q1/6832—Enhancement of hybridisation reaction
    • 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
    • C12Q1/6869—Methods for sequencing

Definitions

  • the present invention relates to genetic engineering More specifically, a method is presented for reducing mispnming during DNA synthesis
  • the present invention relates to primers containing modified nucleosides (e g universal base) which reduce mispnming during cDNA library construction, thereby increasing the proportion of cDNA clones having been primed from the bona fide 3' poly A tail
  • the present invention further relates to the use of the discriminating oligonucleotides of the present invention in other methods such as mRNA purification, PCR-based detection methods and sequencing
  • cDNAs complementary DNAs
  • a full length cDNA allows one to predict transcription initiation start sites, translation initation start sites, deduce certain protein characteristics based on primary ammo acid sequence, predict transcription termination sites, and visually inspect the 5' and 3' untranslated regions for elements which may be involved in post-transcnptional regulation of gene expression
  • the analysis of several complete cDNAs of a given gene enables one to gather information on alternative splicing, alternative promoter usage, and alternative polyadenylation signals - all events known to be important in gene expression regulation
  • the comparison of genomic and cDNA sequence is essential to determine exon-intron structure and document the occurrence of RNA editing - a post-transcnptional regulatory mechanism on which there is little information
  • the cloning of mRNA into cDNA for the purposes of functional studies is a complex, interrelated series of enzyme-catalyzed reactions involving the in vitro synthesis
  • oligo d(T) is utilized, since this is expected to anneal to the 3' poly (A) tail of the mRNA
  • Second strand synthesis is performed utilizing RNAse H, DNA polymerase I, and DNA ligase
  • Nucleic acid hybridization in which a DNA or RNA strand binds to its complement to form a duplex structure is a fundamental process in molecular biology
  • a critical aspect of this process is the specificity of molecular recognition of one strand by the other Sequence differences as subtle as a single base change are sufficient to enable discrimination of short (e g - 14 mer) oligomers, and are frequently used to detect point mutations in genes (Conner et al , 1983, Proc Natl Acad Sci USA 80 278-282 )
  • Molecular discrimination of single point changes using oligonucleotides has been well documented and the underlying thermodynamics well characterized (Ikuta et al , 1987, Nucl Acids Res 15 797-811 , Doktycz et al , 1995, J Biol Chem 270 8439-8445.
  • U S patent 5,438,131 of Bergstrom et al teaches oligonucleotides of at least 10 nucleosides, composed of at least two different bases, and containing at least one universal nucleoside and the use thereof to reduce the element of risk and enhance success in screening DNA libraries
  • the universal base is defined in U S 5,438,131 as being a modified nucleic acid base that can base-pair with its ally, one of the common bases A, T, C and G (as well as U)
  • the aim of the universal base is to reduce degeneracy while still preserving the uniqueness of the probe
  • U S 5,438,131 relates to oligonucleotides containing universal nucleosides at degenerate positions, such that the oligomer allows bonding to unknown bases, enabling the formation of duplexes with ambiguous or unknown nucleic acid sequences
  • U S 5,438,131 relates to oligonucleotides containing universal nucleosides at degenerate positions, such that
  • the invention concerns the identification of primer modifications that can destabilize artifactual duplex formation and decrease the number of mismatches between the primer and its target sequence
  • the invention further concerns the identification of primer modifications that improve the discrimination between the binding thereof to a homopolymeric target sequence (the J ona fide target sequence) as compared to a non-homopolyme ⁇ c target sequence
  • the invention therefore provides oligonucleotides which are better at discriminating between their homopolymeric complementary sequence and a related target sequence
  • the present invention provides assays which can be used (and adapted) to identify oligonucleotide modifications that destabilize mismatches
  • the invention also concerns the development of primers which decrease mispnming events encountered during DNA synthesis More specifically, the invention concerns the development of primers containing at least one modified nucleoside, which decrease the number of internal mispnming events during cDNA generation, thereby improving the efficiency of correct priming from the bona fide 3' poly (A) tail
  • the present invention further relates to universal primers which reduce the proportion of mismatches during genetic engineering methods such as, for example, mRNA purification, 3' RACE, 5' RACE, PCR, sequencing and the like
  • the present invention relates to the incorporation of at least one universal base in an oligonucleotide comprising a homopolymeric stretch in order to reduce mismatches to its homopolymeric target sequence, and thereby generating a modified oligonucleotide
  • the invention concerns more particularly modified oligonucleotides, wherein a homopolyme ⁇ c-stretch of the oligos contains a modification which improves their binding
  • the invention also concerns assays to identify modifications in oligonucleotides which reduce the proportion of mismatches and mispnming events, comprising a random or rational design of modifications of a chosen primer, a hybridization thereof with its target sequence to form a duplex, a synthesis of DNA priming from this duplex and an analysis of the synthesized DNA to assess for the presence of mispnming events, wherein the number of mispnming events produces cDNAs of truncated sizes compared to cDNAs produced by initiation from the bona fide priming site (i e the homopolymeric priming site)
  • a method for destabilizing non-specific duplex formation between an oligonucleotide and a target nucleic acid wherein at least one of the oligonucleotide and target nucleic acid comprises a homopolymeric sequence
  • the method comprising an incubation of the target nucleic acid with a modified oligonucleotide, wherein the modified oligonucleotide includes a modification which decreases or abrogates hydrogen bonding between same and nonspecific target sequences and thus enables a discrimination between a Jbona fide duplex formation and an artifactual one, under the conditions of hybridization used
  • the target nucleic acid is a homopolymeric sequence
  • a method for increasing the proportion of full length cDNA clones in a library comprising a use of a modified oligo d(T) primer during first strand synthesis, wherein the modification decreases or abrogates hydrogen bonding between the modified oligo d(T) primer and a non-specific target sequence, thereby increasing the proportion of full length cDNA clones
  • a method for reducing mispnming events during DNA synthesis comprising a use of a modified oligonucleotide to prime the DNA synthesis, wherein the modification decreases or abrogates hydrogen bonding between the modified primer and a non-specific target sequence, thereby reducing mispnming events
  • modified oligonucleotide primers that destabilize non-specific duplex formation and reduce mispnming during DNA synthesis
  • the present invention provides the means to destabilize mispai ⁇ ng of an oligonucleotide or primer to a
  • oligo d(T)*Z primer an oligo d(T) primer in which two of the thymine bases are substituted by 3-n ⁇ tropyrrole
  • the instant invention is not so limited
  • the position of the modified bases within the exampl fied oligonucleotide, oligo d(T) primer can be altered (Fig 2C) without changing the discrimination between primer and either complementary template or partially complementary template
  • Guo et al (1997, supra) have changed the position of 2 universal nucleosides within a given heteropolymenc oligonucleotide and shown that in many cases increased discrimination between perfect matched template and mismatched template is maintained
  • the instant invention extends to any homopolymeric-stretch-contaming oligonucleotide (or any oligonucleotide designed to bind to a homopolymeric target sequence) such as an oligo d(T) primer containing modified nucleosides at
  • the present invention should not be limited to the modifications of oligonucleotides with 3-n ⁇ tropyrrole, since other universal bases are well known in the art Indeed, in addition to 3-n ⁇ tropyrrole, a number of universal nucleosides have been synthesized and characterized (Millican et al 1984, supra, Inone et al , 1985, supra, Fukada et al , 1986, supra, Seela et al ,1986, supra, Eritja et al , 1986, supra, Habener et al , 1988, supra, Lin et al , 1989, supra, Francois et al , 1990, supra, Brown et al , 1991 , supra) Other examples of universal bases can be found at www Synthegen com/products/bases html Thus, the present invention covers any homopolyme ⁇ c-stretch-containing oligo (e g oligo d(T) primer), containing at least one universal nucleoside which
  • a non-limiting example of an alternative use of this technology is in mRNA purification, by replacing oligo d(T) affinity matrixes currently employed with modified oligo d(T) according to the instant invention
  • An oligo d(T)*Z affinity matrix would perform the same task, except that binding to internal A-nch stretches would be minimized and could result in a purification method with a higher stringency than currently employed
  • This matrix could provide a better selection between eukaryotic mRNA and contaminating mycoplasmic RNA (which is A-T rich) Since mycoplasms often contaminate tissue culture cell lines, co-purification of mycoplasma RNA with eukaryotic mRNA on oligo d(T) column can produce cDNA libraires contaminated with mycoplasma clones
  • RNA sequence of a particular RNA must be interogated Reverse transcnptase (RT), in combination with PCR, can be used to amplify a given region on an RNA template
  • RT Reverse transcnptase
  • oligo d(T)»Z primer in the RT reaction would ensure that the 3' end of the mRNA is represented on the cDNA template
  • the present invention can also be incorporated into current 3' RACE (Rapid Amplification of cDNA Ends) protocols, designed to obtain the 3' end of a given clone
  • first strand synthesis is followed by homopolymeric tailing of the products utilizing terminal deoxynucleotidyl transferase
  • dGTP can be utilized to add a homopolymeric stretch of G's at the 5' end of the cDNA
  • the DNA polymerase utilized in second strand synthesis can take advantage of this G-stretch by priming from an oligo d(C) primer annealed to the G-stretch positioned at the 5' end
  • This procedure has the advantage of maintaining the sequence at the 5' terminal end of the cDNA, and is also used in 5' RACE strategies to identify the 5' end of mRNAs (Frohman et al , 1988, Proc Natl Acad Sci USA 85 8998-9002, Loh et al , 1989, Science 243 217-220) (Fig 5)
  • 5' untranslated regions of mRNAs are usually GC rich,
  • modified oligonucleotides wherein the modified oligo comprises a homopolymeric stretch containing at least one universal nucleoside (or other non-specific duplex destabilizing modifications), to achieve increased discrimination between a target site (or several target sites) of interest when generating a specific product or a set of products (for example use of an oligo d(T) primer to prime DNA synthesis from the A-nch stretch of Alu repeats in humans) Since these products can be developed to be used as genetic markers (by identifying polymorphisms residing with the sequence of the product), changing the specificity of targeting by altering the specificity of the oligo d(T) primer, could result in a more consistent representation of the final PCR products
  • the present invention thus further relates to the use of universal oligonucleotides or other modified oligonucleotides, during PCR amplification DEFINITIONS
  • Nucleotide sequences are presented herein by single strand, in the 5' to 3' direction, from left to right, using the one letter nucleotide symbols as commonly used in the art and in accordance with the recommendations of the IUPAC-IUB Biochemical Nomenclature Commission
  • nucleic acids are routinely used recombinant DNA (rDNA) technology terms Nevertheless, definitions of selected examples of such rDNA terms are provided for clarity and consistency For certainty, it is emphasized that the present invention finds utility with nucleic acids in general
  • nucleic acids which can be used in accordance with the teachings of the present invention include that from eukaryotic cells such as that of animal cells, plant cells, or microorganisms as well as that from prokaryotic cells
  • homopolymeric sequences refers to a sequence composed of a single type of nucleotide base (adenosine A, cytosine C, guanine G, thymine T, uracil U) or of a less common base (non- limiting examples including inosine, I, and pseudoundine, ⁇ )
  • nucleic acid molecule refers to a polymer of nucleotides
  • Non-limiting examples thereof include DNA (e g genomic DNA, cDNA) and RNA molecules (e g mRNA)
  • the nucleic acid molecule can be obtained by cloning techniques or synthesized DNA can be double-stranded or single-stranded (coding strand or non-coding strand [antisense])
  • recombinant DNA refers to a DNA molecule resulting from the joining of DNA segments This is often referred to as genetic engineering
  • amplification pair refers herein to a pair of oligonucleotides (oligos) of the present invention, which are selected to be used together in amplifying a selected nucleic acid sequence by one of a number of types of amplification processes, preferably a polymerase chain reaction Other types of amplification processes include ligase chain reaction, strand displacement amplification, or nucleic acid sequence-based amplification, as explained in greater detail below As commonly known in the art, the oligonucleotides are designed to bind to a complementary sequence under selected conditions
  • the nucleic acid e g DNA or RNA
  • the nucleic acid for practicing the present invention may be obtained according to well known methods
  • Oligonucleotides or “oligos” or “primers” define a nucleic acid molecule composed of nucleotides (ribo or deoxynbonucleotides)
  • Oligonucleotide probes or primers of the present invention may be of any suitable length, depending on the particular assay format and the particular needs and targeted genomes employed In general, the oligonucleotide probes or primers are at least 10 nucleotides in length, preferably below 50 nucleotides Preferably, the oligos or primers have lengths between 15 and 40 nucleotides, more preferably between 20 to 30 nucleotides Of course, the probes or primers of the present invention may be adapted to be especially suited to a chosen nucleic acid amplification system As commonly known in the art, the oligonucleotide probes and primers can be designed by taking into consideration the melting point of hydnzidation thereof with its targeted sequence (see below and in Sambrook et al
  • oligonucleotide or "DNA” molecule or sequence refers to a molecule comprised of the deoxynbonucleotides adenine (A), guanine (G), thymine (T) and/or cytosine (C), in a double-stranded form, and comprises or includes a "regulatory element” according to the present invention, as the term is defined herein
  • oligonucleotide or “DNA” can be found in linear DNA molecules or fragments, viruses, plasmids, vectors, chromosomes or synthetically derived DNA As used herein, particular double-stranded DNA sequences may be described according to the normal convention of giving only the sequence in the 5' to 3' direction
  • Probes and oligonucleotides of the invention can be utilized with naturally occurring sugar-phosphate backbones as well as modified backbones including phosphorothioates, dithionates, alkyl phosphonates and ⁇ -nucleotides and the like Modified sugar-phosphate backbones are generally taught by Miller, 1988, Ann Reports Med Chem 23 295 and Moran et al , 1987, Nucleic acid molecule Acids Res , 14 5019 Probes of the invention can be constructed of either nbonucleic acid (RNA) or deoxy ⁇ bonucleic acid (DNA), and preferably of DNA General teachings on the synthesis of oligonucleotides and substituents and modifications thereof can be found for example in US 5,438, 131 The selection of the best suited synthesis pathway of an oligonucleotide and of the appropriate modifications, and substituents to be used, may be selected accordingly by the person of ordinary skill to which the instant invention pertains The modified oligonucleotides of
  • Nucleic acid hybridization refers generally to the hybridization of two single-stranded nucleic acid molecules having complementary base sequences, which under appropriate conditions will form a thermodynamically favored double-stranded structure
  • hybridization conditions can be found in the two laboratory manuals referred above (Sambrook et al , 1989, supra and Ausubel et al , 1989, supra) and are commonly known in the art
  • a hybridization to a nitrocellulose filter as for example in the well known Southern blotting procedure, a nitrocellulose filter can be incubated overnight at 65°C with a labeled probe in a solution containing 50% formamide, high salt (5 x SSC or 5 x SSPE), 5 x Denhardt's solution, 1 % SDS, and 100 ⁇ g/ml denatured carrier DNA (e g salmon sperm DNA)
  • the non-specifically binding probe can then be washed off the filter by several washes in 0 2 x SSC/0 1 % SDS
  • probes can be used include Southern blots (DNA detection), dot or slot blots (DNA, RNA), and Northern blots (RNA detection)
  • Probes or oligonucleotides can be labeled according to numerous well known methods (Sambrook et al , 1989, supra)
  • Non-limiting examples of labels include 3 H, 14 C, 32 P, and 35 S
  • Non-limiting examples of detectable markers include ligands, fluorophores, chemiluminescent agents, enzymes, and antibodies
  • Other detectable markers for use with probes, which can enable an increase in sensitivity of the method of the invention, include biotin and radionucleotides It will become evident to the person of ordinary skill that the choice of a particular label dictates the manner in which it is bound to the probe
  • radioactive nucleotides can be incorporated into probes of the invention by several methods Non-limiting examples thereof include kinasing the 5' ends of the probes using gamma 32 P ATP and polynucleotide kinase, using the Klenow fragment of Pol I of E coll or reverse transcnptase in the presence of radioactive dNTP (e g uniformly labeled DNA probe using random oligonucleotide primers in low-melt gels), using the SP6/T7 system to transcribe a DNA segment in the presence of one or more radioactive NTP, and the like
  • radioactive dNTP e g uniformly labeled DNA probe using random oligonucleotide primers in low-melt gels
  • Amplification of a selected, or target, nucleic acid sequence may be carried out by a number of suitable methods See generally Kwoh et al , 1990, Am Biotechnol Lab 8 14-25 Numerous amplification techniques have been described and can be readily adapted to suit particular needs of a person of ordinary skill Non-limiting examples of amplification techniques include polymerase chain reaction (PCR), hgase chain reaction (LCR), strand displacement amplification (SDA), transcription-based amplification, the Q ⁇ replicase system and NASBA (Kwoh et al , 1989, Proc Natl Acad Sci USA 86, 1173-1177, Lizardi et al , 1988, BioTechnology 6 1197-1202, Malek et al , 1994, Methods Mol Biol , 28 253-260, and Sambrook et al , 1989, supra)
  • PCR Polymerase chain reaction
  • PCR Polymerase chain reaction
  • Ligase chain reaction is carried out in accordance with known techniques (Weiss, 1991 , Science 254 1292) Adaptation of the protocol to meet the desired needs can be carried out by a person of ordinary skill Strand displacement amplification (SDA) is also carried out in accordance with known techniques or adaptations thereof to meet the particular needs (Walker et al , 1992, Proc Natl Acad Sci USA 89 392-396, and ibid , 1992, Nucleic Acids Res 20 1691-1696)
  • SDA Strand displacement amplification
  • the term "gene” is well known in the art and relates to a nucleic acid sequence defining a single protein or polypeptide
  • a "structural gene” defines a DNA sequence which is transcribed into RNA and translated into a protein having a specific ammo acid sequence thereby giving rise to a specific polypeptide or protein It will be readily recognized by the person of ordinary skill, that the nucleic acid sequence of the present invention can be incorporated into anyone of numerous established kit formats which are
  • vector is commonly known in the art and defines a plasmid DNA, phage DNA, viral DNA and the like, which can serve as a DNA vehicle into which DNA of the present invention can be cloned
  • vectors Numerous types of vectors exist and are well known in the art
  • allele defines an alternative form of a gene which occupies a given locus on a chromosome
  • a “mutation” is a detectable change in the genetic material which can be transmitted to a daughter cell
  • a mutation can be, for example, a detectable change in one or more deoxynbonucleotide
  • nucleotides can be added, deleted, substituted for, inverted, or transposed to a new position Spontaneous mutations and experimentally induced mutations exist
  • the result of a mutations of nucleic acid molecule is a mutant nucleic acid molecule
  • a mutant polypeptide can be encoded from this mutant nucleic acid molecule
  • purified refers to a molecule having been separated from a cellular component
  • a purified protein has been purified to a level not found in nature
  • a “substantially pure” molecule is a molecule that is lacking in most other cellular components
  • a compartmentalized kit in accordance with the present invention includes any kit in which reagents are contained in separate containers
  • Such containers include small glass containers, plastic containers or strips of plastic or paper
  • Such containers allow the efficient transfer of reagents from one compartment to another compartment such that the samples and reagents are not cross-contaminated and the agents or solutions of each container can be added in a quantitative fashion from one compartment to another
  • Such containers will include a container which will accept the test sample (DNA, RNA or cells), a container which contains the primers used in the assay, containers which contain enzymes, containers which contain wash reagents, and containers which contain the reagents used to detect or isolate the extension products
  • cDNA cloning kits could be adapted by inserting thereinto the primers of the present invention
  • Figure 1 shows an example of the steps involved in generating cDNA libraries from mRNA
  • a primer from which the reverse transcnptase (RT) can prime
  • an oligo d(T) primer is used because it anneals to the 3' poly (A) tail of the eukaryotic mRNAs
  • a homopolymeric stretch of nucleoside 5'-monophosphates can be added to the 3' end of the mRNA
  • poly (A) polymerase can be used to add a poly (A) tail to mRNAs which lack one
  • An oligonucleotide which contains complementary nucleotides e g oligo d(T)
  • Figure 2A shows a hybridization of oligo d(T) 15 primer to the bona fide poly (A) tail of an mRNA (right) or to an internal A-nch stretch (left) within the mRNA by conventional oligo d(T) primer used in current cDNA library construction
  • the length of the primer used can differ, and the two 3' most nucleotides are sometimes (A,C,G,T) and (A,G,C) to "lock" the oligonucleotide in place at the junction of the body of the mRNA and the poly (A) tail, neither of these modifications prevent the misannealing of the oligo d(T) primer to internal A-nch stretches
  • Figure 2B shows the chemical structure of 3- nitropyrrole
  • Figure 2C shows the structure of oligo d(T)»Z primer
  • Figure 2D shows the expected discrimination between the poly (A) tail (right) and internal A-nch stretches (
  • Figure 3A shows the structure of the elF-4GII cDNA construct used to analyze mispnming at the 3' end The location of four internal A-nch sequences are shown - all of which generated 3 1 truncated clones when elF-4GII was isolated from a cDNA library The plasmid was linearized with Asp 718 and T7 RNA polymerase used to generate a -2400 nt 3 H-test transcript
  • Figure 3B shows the integrity of the in vitro generated transcript following fractionation on a formaldehyde 1 2% agarose gel, treatment with EN 3 HANCE, and autoradiography of the dried gel
  • Figure 3C shows the alkaline agarose analysis of RT products generated by priming synthesis with oligo d(T) (lane 1) or oligo d(T)*Z (lane 2) using MMLV RT Complementary DNA was labeled with ⁇ - 32 P- dCTP The position of migration of truncated products are indicated by a
  • Figure 4A shows the structure of elF-4GII construct used to demonstrate mispnming at the 3' end
  • the location of five oligonucleotides (a, b, c, d, e) used in the hybridization assay to map the sites of 3' mispnming by oligo d(T) are shown
  • the nucleotide targets of the oligonucleotides on elF-4GII are shown.
  • Figure 4B shows the Southern blot of the alkaline agarose gel of RT products generated by priming synthesis with either oligo d(T) or ohgo d(T)*Z
  • Marker lane refers to the 1 kb size ladder from GIBCO and sizes (in bp) are indicated to the left of the diagram
  • elF-4GII DNA refers to a DNA fragment of elF-4G!l used as a positive control for DNA hybridizations
  • Oligonucleotides used as probes on each blot are indicated below each panel
  • the asterisks on the left denotes the cDNA product obtained by priming at the correct poly (A) site
  • the filled circle denotes the cDNA product obtained by priming from the A- rich stretch between nucleotides 5550-5575, whereas the arrow denoted the cDNA obtained by priming from nucleotides 5085-5120
  • Figure 5 shows an example illustrating mispnming events at the 5' end of cDNAs during cDNA library construction of 5' RACE analysis to extend the sequence of known genes
  • Figure 6 shows an autoradiograph following ohgo d(T) primed first strand synthesis on elF-4GII mRNA
  • Lane 2 ohgo d(T) control, lane 3 ohgo d(T)*Z, and lane 4 ohgo d(T)*l
  • a molecular mass standard ladder is shown in lane 1
  • Mispnming event are common in Rapid Amplification of cDNA ends (RACE)
  • RACE Rapid Amplification of cDNA ends
  • FIG. 5 An example of mispnming at the 5' end of cDNAs during 5' RACE analysis is shown in Figure 5
  • Such mispnming events could be resolved by incorporating a universal nucleoside into the ohgo d(C) primer to increase the discrimination between the homologous target (e g - the 5' end G tail) and an internal G- ⁇ ch sequence It is expected that incorporation of at least one universal base (e g 3-n ⁇ tropyrrole) in the homopolymeric ohgo d(C) primer should significantly reduce such mispnming
  • the o go d(T) primer was modified by inserting thereinto deoxynucleotide deoxyinosme (I)
  • oligonucleotide [called o go d(T)*l] having the sequence 5TTTTTTTI*TTTTTTTTTI * TTTTT3' was thus synthesized (McGill University Sheldon Biotechnology Center), where I* represents the position where the 2'deoxy ⁇ nos ⁇ ne was incorporated into the oligonucleotide
  • Reverse transcription reactions were performed on in vitro generated elF-4G mRNA templates (1 ⁇ g) with Superscript IITM (LifeTechnologies) under conditions recommended by LifeTechnologies
  • Oligonucleotide primers that were utilized to prime the first strand synthesis were 0 1 ⁇ g of either Ohgo d(T) 15 , o go d(T)»Z, or oligo d(T)»l
  • the radioisotope ⁇ - 32 P-dCTP was used as a tracer to monitor the quality of the cDNA product Following the generation of cDNA products at 42°C for 1 hr, the mixture was extracted with
  • FIG. 6 An photograph of the autoradiograph is presented in Figure 6 A molecular mass standard ladder is shown in lane 1 (purchased from LifeTechnologies) The cDNA product obtained by priming with ohgo d(T) 15 is shown in lane 2 Clearly, the major cDNA product is shorter than full-length and arises due to internal mispnming at an internal A-nch site As shown previously, priming with ohgo d(T)*Z is able to correct the mispnming phenomenon, and in this particular experiment over 50% of the cDNA is correctly primed from the poly (A) tail of the mRNA (lane 3, full-length product indicated with an arrow) Priming the cDNA reaction with ohgo d(T)*l also efficiently corrected the mispnming reaction observed with o go d(T) 15 primer and resulted in a significant proportion of cDNAs being full-length (lane 4 full length product indicated with an arrow)

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Abstract

L'invention constitue la preuve qu'en modifiant une extension homopolymère dans un oligonucléotide, ou amorce, on améliore la discrimination de la liaison dudit nucléotide ou de ladite amorce modifié(e) avec sa séquence cible homopolymère complémentaire par rapport à une liaison avec une séquence non homopolymère. Plus spécifiquement, on a utilisé une amorce oligo d(T), dans laquelle deux des bases thymine ont été substituées par un 3-nitropyrrole, pour réaliser une expérience de synthèse d'ADNc amorcée par un poly A prouvant que la discrimination de l'amorçage de la synthèse d'ADNc à partir de la séquence poly A bona fide est supérieure à celle obtenue avec des séquences internes riches en A. L'invention concerne des modifications de séquences homopolymères dans les oligos, diminuant la capacité de liaison par pontage, en général, puisqu'il a été démontré que d'autres modifications, telles que la substitution d'une amorce oligo d(T) par la 2' désoxy-inosine, améliorent également la discrimination de la liaison avec une queue poly A bona fide par rapport à une liaison avec des séquences riches en A. L'invention concerne donc des amorces universelles qui diminuent les erreurs d'amorçage lors de la création d'une banque d'ADNc, ce qui augmente la proportion de clones d'ADN amorcés à partir d'une queue 3' poly A bona fide. Elle concerne aussi l'utilisation des oligonucléotides discriminants de la présente invention dans d'autres techniques telles que la purification de l'ARNm, les méthodes de détection basées sur la PCR et le séquençage.
EP99947148A 1998-10-07 1999-10-06 Amorces oligonucleotidiques destabilisant la formation de duplex non specifiques et leurs utilisations Withdrawn EP1117826A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
CA2246623 1998-10-07
CA 2246623 CA2246623A1 (fr) 1998-10-07 1998-10-07 Amorces oligonucleotidiques qui destabilisent la formation de double brin non specifique et reduisent les risques de choisir les mauvaises amorces pendant la construction de la banque d'adnc et leurs utilisations
PCT/CA1999/000933 WO2000020630A1 (fr) 1998-10-07 1999-10-06 Amorces oligonucleotidiques destabilisant la formation de duplex non specifiques et leurs utilisations

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EP1117826A1 true EP1117826A1 (fr) 2001-07-25

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JP (1) JP2002532063A (fr)
AU (1) AU6073299A (fr)
CA (1) CA2246623A1 (fr)
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WO2002042490A1 (fr) * 2000-11-24 2002-05-30 Asper OÜ Procede pour optimiser les sequences d'acides nucleiques synthetiques
US9261460B2 (en) * 2002-03-12 2016-02-16 Enzo Life Sciences, Inc. Real-time nucleic acid detection processes and compositions
US20030175749A1 (en) 2001-12-08 2003-09-18 Jong-Yoon Chun Annealing control primer and its uses
ES2257895B1 (es) * 2003-05-30 2008-03-16 Consejo Sup. De Invest. Cientificas Procedimiento de optimizacion de oligonucleotidos y de las condiciones de amplificacion mediante pcr para race (5' and 3'-rapid amplification of cdna ends).
WO2006076650A2 (fr) * 2005-01-12 2006-07-20 Applera Corporation Compositions, procedes et kits pour l'amplification selective d'acides nucleiques
JP2012000044A (ja) * 2010-06-16 2012-01-05 Hitachi Ltd 大規模並列核酸分析方法
GB2497510A (en) * 2011-11-10 2013-06-19 Harry Cuppens Methods for determining mononucleotide sequence repeats

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US5438131A (en) * 1992-09-16 1995-08-01 Bergstrom; Donald E. 3-nitropyrrole nucleoside
JP4494529B2 (ja) * 1995-11-16 2010-06-30 ダコ デンマーク アクティーゼルスカブ 真核系サンプル中の特異的核酸配列を検出するためのｉｎ ｓｉｔｕハイブリダイゼーション

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Title
See references of WO0020630A1 *

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CA2246623A1 (fr) 2000-04-07
WO2000020630A1 (fr) 2000-04-13
JP2002532063A (ja) 2002-10-02

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