WO2000020630A1

WO2000020630A1 - Oligonucleotide primers that destabilize non-specific duplex formation and uses thereof

Info

Publication number: WO2000020630A1
Application number: PCT/CA1999/000933
Authority: WO
Inventors: Jerry Pelletier; Manjula Das
Original assignee: Mcgill University
Priority date: 1998-10-07
Filing date: 1999-10-06
Publication date: 2000-04-13
Also published as: JP2002532063A; EP1117826A1; CA2246623A1; AU6073299A

Abstract

The present invention relates to the demonstration that a modification of a homopolymeric stretch in an oligonucleotide, or primer, improves the discrimination for binding of such a modified oligonucleotide or primer to its complementary homopolymeric target sequence, as compared to a non-homopolymeric sequence. More specifically, an oligo d(T) primer having two of the thymine bases substituted by 3-nitropyrrole were used in a poly A primed cDNA synthesis experiment to demonstrate an improvement in discrimination between the priming of cDNA synthesis from bona fide poly A sequence as compared to internal A-rich sequences. The present invention relates to modifications of homopolymeric sequences in oligos, decreasing the ridging bonding capacity, in general, since other modifications, such as an oligo d(T) primer substituted with 2' deoxyinosine was also shown to improve the discrimination between the binding to a bona fide poly A tail as compared to A-rich sequences. The present invention thus relates to universal primers which reduce mispriming 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.

Description

TITLE OF THE INVENTION

OLIGONUCLEOTIDE PRIMERS THAT DESTABILIZE NONSPECIFIC DUPLEX FORMATION AND USES THEREOF

FIELD OF THE INVENTION

The present invention relates to genetic engineering More specifically, a method is presented for reducing mispnming during DNA synthesis In particular, 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

BACKGROUND OF THE INVENTION

The isolation and rapid mapping of complementary DNAs (cDNAs) is central to characterizing the information that is of significant biological relevance in the genome of an organism 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 In addition, 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 of a DNA copy of mRNA, its subsequent conversion to duplex cDNA, and insertion into an appropriate prokaryotic vector The procedure may involve the following series of steps (outlined in Fig 1)

1) Isolation of high quality mRNA from the tissue or cell line of interest

2) Annealing of a DNA oligonucleotide, either a mixture of oligonucleotides of random sequence or an oligo d(T) primer, to the mRNA When full-length cDNAs are required, oligo d(T) is utilized, since this is expected to anneal to the 3' poly (A) tail of the mRNA

3) Reverse transcπptase is then utilized to prime from the DNA primer and copy the RNA template into cDNA

4) Second strand synthesis is performed utilizing RNAse H, DNA polymerase I, and DNA ligase

5) The ends of the cDNAs are polished, prepared for cloning, and the cDNAs are introduced into an appropriate cloning vector

Although a number of different approaches can be used to generate cDNA libraries, they suffer from several major problems, often making the isolation of a complete cDNA an arduous task The cloning of incomplete cDNAs is widespread, resulting in only partial characterization of mRNA transcripts and significantly increasing the cost and amount of work required to obtain a full-length copy of the cDNA of interest One major reason why many clones in current cDNA libraries are not full-length is due to mispnming of the oligo d(T) primer (de Fatima Bonaldo et al , 1996, Genome Res 6 791-806) Many eukaryotic mRNAs contain regions of A-nch stretches within their sequence Thus oligo d(T) primers can anneal to these internal A-nch stretches When reverse transcnptase primes from these internal sites, sequence information from the 3' end of the mRNA is lost during the cDNA cloning process (Fig 1) Although the genetic code of most organisms is composed of ~ 50% guanosine + cytosine residues and 50% of adenosine + thymidine residues, there are well known examples of organisms whose genetic code deviates from this ratio For example, the genome of the parasite responsible for malaria transmission, Plasmodium falciparum, has a genome of >80% adenosine + thymidine residues (Weber, J L , 1987, Gene 52 103-109) This implies that cDNA libraries derived from this organism will contain many truncated, less- than-full-length clones, due to mispnming of the oligo d(T) primer during first strand synthesis Mispnming is thus a serious hindrance to gene discovery and characterization in general, and more acutely for certain organisms These technical limitations imply that a set of products of variable length are often generated during first strand synthesis Consequently, a number of truncated clones may be present in any given library Given these cloning complications, interpretations about gene structure are sometimes misleading and cDNA cloning is often inefficient, costly and time-consuming - often requiring the sampling of several different libraries

The actual procedure for generating cDNA libraries has not extensively deviated from the original method of Gubler et al , 1983, Gene 25 263-269 Because of the frequent generation of products of variable length during first strand synthesis, a number of truncated clones will be present in libraries for any given gene Priming from the poly (A) tract of mRNAs with oligo d(T) is necessary to obtain a copy of the entire 3' untranslated region However, it is the experience of many laboratories screening cDNA libraries, that a significant proportion of clones do not have a bona fide 3' end, due to misannea ng of the oligo d(T) primer to internal A-nch sites Indeed, cDNAs with 3' truncations are estimated to occur at frequencies of 10-15% in some libraires (de Fatima Bonaldo et al , 1996, supra) Such clones are easily recognized by the absence of a bona fide polyadenylation signal sequence -20 nucleotides upstream of the oligo (dA) tail of the cDNA If enhanced discrimination could be achieved between annealing to the bona fide poly (A) tail versus internal A-πch sequences by the Reverse Transcnptase primer, then the frequency of this "mispnming artifact" would be significantly reduced

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. Southern et al , 1994, Nucl Acids Res 22 1368-1373, Saiki et al , 1989, Proc Natl Acad Sci USA 86 6230-6234) However, in many cases, the stability difference between a perfectly matched complement (e g - between a poly (A) tail and oligo d(T)₁₅) and a complement mismatched at only one base (e g - between AAAAAAATAAAAAAA and oligo d(T)₁₅) can be quite small, corresponding to as little as 0 5°C difference in their duplex melting temperature (T_ms) (Fig 2) The longer the oligomer of interest (e g an oligo d(T)₂₀ primer versus and oligo d(T)₁₅ primer) the smaller the effect of a single-base mismatch on overall duplex stability This limitation in hybridization is the major reason why oligo d(T) primers often hybridize to internal A-nch sequences on mRNA templates during cDNA library construction, and consequently why a large number of clones in such libraries do not contain the bona fide 3' end Guo et al (1997 , Nature Biotech 15 331-335) have recently shown that increased discrimination of single nucleotide mismatches by oligonucleotides can be achieved by introducing artifical mismatches into the probe oligonucleotide using the base analog 3-nιtropyrrole This base analog acts as a universal nucleoside that hydrogen bonds minimally with all four bases without steric disruption of the DNA duplex (Nichols et al , 1994, Nature 369 492-493) Since hydrogen bonding between bases of two complementary strands of DNA is the major thermodynamic force responsible for maintaining the integrity of a double stranded DNA duplex, base substitutions with analogs with lessened hydrogen bond capacity can function as universal nucleosides (Nichols et al , 1994, supra) A number of different nucleoside analogs have been developed which function in this fashion (Millican et al , 1984, Nucl Acids Res 12 7435-7453, Inone et al , 1985, Nucl Acids Res 13 7119-7128, Fukada et al , 1986, Naturforsch B 41 1571-1579, Seela et al , 1986, Nucl Acids Res 14 1825-1844, Eritja et al , 1986, Nucl Acids Res 14 8135-8153, Habener et al , 1988, Proc Natl Acad Sci USA 85 1735-1739, Lin et al , 1989, Nucl Acids Res H 10373-10383, Francois et al , 1990, Tetrahedron Lett 316347-6350, Brown et al , 1991 , Carbohydrate Res 216 129-139 ) Guo et al (1997, supra) have shown that the introduction of universal analogues into heteropolymeπc oligonucleotides during their synthesis, increases the thermal stability (ΔTJ of hybrids formed between an ohgonucleotide with the universal nucleoside and normal and single-nucleotide variant DNA targets by as much as 200%, as compared to hybrids formed between a wild-type ohgonucleotide and normal or single-nucleotide variant DNA targets

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 A variety of compounds have been investigated as universal bases and a number of them are described in U S 5,438,131 In a preferred embodiment, 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 In a particularly preferred embodiment, U S 5,438,131 relates to 3-nιtropyrrole nucleoside as the universal nucleoside U S 5,438,131 thus relates to the use of universal nucleosides in order to stabilize duplex formation between heteropolymers of oligonucleotides and a target nucleic acid

In view of the technical limitations of current methods of cDNA synthesis, there remains a need to destabilize artefactual duplex formation to increase the discrimination between specific and non-specific duplexes There also remains a need to provide the means to reduce mismatches in general, and more particularly to reduce mispnming during DNA synthesis, cDNA library construction, and PCR applications The present invention seeks to meet these and other needs

The present description refers to a number of documents, the content of which is herein incorporated by reference in their entirety

SUMMARY OF THE INVENTION

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 In one embodiment, 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-homopolymeπc target sequence The invention therefore provides oligonucleotides which are better at discriminating between their homopolymeric complementary sequence and a related target sequence In addition, 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 In a particularly preferred embodiment, 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 homopolymeπc-stretch of the oligos contains a modification which improves their binding to their target sequence More specifically, the present invention relates to primers or oligos incorporating at least one 3-nιtropyrrole modification in the homopolymeric stretch

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)

In accordance with the present invention, there is therefore provided 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 In accordance with a preferred embodiment of the present invention, the target nucleic acid is a homopolymeric sequence

In accordance with the present invention, there is also provided 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

In accordance with another aspect of the present invention, there is provided 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

In accordance with yet another aspect of the present invention, there is provided modified oligonucleotide primers that destabilize non-specific duplex formation and reduce mispnming during DNA synthesis

While the method of the instant invention is demonstrated during first strand cDNA synthesis to improve the quality of the cDNA population by reducing the number of clones containing aberrant 3' ends due to oligo d(T) mispaiπng, and more specifically using the elF-4GII mRNA template, the present invention, which has broad utility, is not so limited Although 3' mispaiπng is a general problem encountered when generating cDNA libraries from a number of organisms, this problem can be particularly exacerbated when generating cDNA libraries from organisms that have A rich genomes, since the number of internal A-nch stretches will be higher in genes from these organisms This type of incomplete A-tract is expected to misanneal to oligo d(T) and produce truncated cDNAs during library construction An example of such an organism is Plasmodium falciparum, the parasite responsible for malaria transmission by mosquitos Thus, the present invention provides the means to destabilize mispaiπng of an oligonucleotide or primer to a non-targeted or non-specific nucleic acid sequence from any organism or nucleic acid sequence-containing entity, thereby increasing the proportion of duplexes formed between the oligonucleotide or primer and its proper targeted sequence In one preferred embodiment of the present invention, the modified primer comprises an essentially homopolymeric stretch of nucleotides (including a modification) which targets its complementary homopolymeric sequence

While the instant invention is demonstrated using an oligo d(T)*Z primer (an oligo d(T) primer in which two of the thymine bases are substituted by 3-nιtropyrrole), the instant invention is not so limited For example, 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 Indeed, 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 Thus, 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 any position, provided that such modification maintains the discriminating ability of the oligonucleotide under suitable assay conditions It should be clear to the person of ordinary skill that the present invention further provides the means to assess whether the modifications alter this discriminating activity of the oligonucleotide It should also be clear that any type of homopolymeπc- complementary sequence duplex formation could be improved by the instant invention In a broad sense therefore, the present invention provides the means and methods to generate oligos or primers with improved discrimination to their complementary homopolymeric sequence compared to non-complementary sequence It should be clear to a person of ordinary skill that the present invention has broad implications since it demonstrates that a modification which results in destabilization of a duplex (examp fied with oligo d(T), having 2 substitutions, and its poly A target sequence), significantly decreases the proportion of mismatches and of mispnming events Hence, it is expected that other types of destabilization of the hydrogen bonds between an oligonucleotide and its target sequence would have the same effect Non-limiting examples of modifications of the oligonucleotide which would result in such a destabilization of the duplex formation, include modifications which reduce or abrogate hydrogen bonding Non-limiting more specific examples include known base modifications, base analogs (e g inosine, as exemplified hereinbeiow), universal bases, and partial mismatches Of course it will be understood that such modifications should not favor duplex formation with a non-desired target sequence

It should also be understood that the different modifications of the oligonucleotides encompassed by the present invention can be adapted by the person of ordinary skill to suit particular utilities (e g mRNA purification, sequencing)

The present invention should not be limited to the modifications of oligonucleotides with 3-nιtropyrrole, since other universal bases are well known in the art Indeed, in addition to 3-nιtropyrrole, 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 homopolymeπc-stretch-containing oligo (e g oligo d(T) primer), containing at least one universal nucleoside which allows for enhanced discrimination when hybridizing to perfect versus mismatched templates In addition, the demonstration that a base analog, such as inosine, inserted into the homopolymeric stretch also enhances discrimination between the target sequence and a mismatched sequence, shows that the present invention covers any homopolymeric-stretch-compπsing oligo containing at least one modified base which reduces or abrogates hydrogen bonding in the sequence which is complementary to the targeted sequence

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, for example, 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

Often, the 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 The use of oligo d(T)»Z as primer in the RT reaction would ensure that the 3' end of the mRNA is represented on the cDNA template Thus, 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

In some cloning protocols, first strand synthesis is followed by homopolymeric tailing of the products utilizing terminal deoxynucleotidyl transferase For example, dGTP can be utilized to add a homopolymeric stretch of G's at the 5' end of the cDNA Thus 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) One drawback of this approach however is that, since 5' untranslated regions of mRNAs are usually GC rich, the oligo d(C) can mispπme from internal G-nch regions, producing less than full-length cDNAs It is expected that the incorporation of universal nucleosides into such homopolymeπc-stretch-containing primers to generate the modified oligos or primers of the present invention will increase the specificity of binding and generate cDNAs which terminate at the bona fide 5' end Thus, the present invention further relates to cloning procedures or RACE protocols involving priming of second strand synthesis from a homopolymeric tail

It may be desirable in some PCR protocols to utilize modified oligonucleotides according to the present invention, 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

Unless defined otherwise, the scientific and technological terms and nomenclature used herein have the same meaning as commonly understood by a person of ordinary skill to which this invention pertains Generally, the procedures for cell cultures, infection, molecular biology methods and the like are common methods used in the art Such standard techniques can be found in reference manuals such as for example Sambrook et al (1989, Molecular Cloning - A Laboratory Manual, Cold Spring Harbor Laboratories) and Ausubel et al (1994, Current Protocols in Molecular Biology, Wiley, New York) The present description refers to a number of 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 Non-limiting examples of 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

As used herein, the term "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, Ψ)

As used herein, "nucleic acid molecule", "nucleic acid sequence" or "sequence" refer 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])

The term "recombinant DNA" as known in the art refers to a DNA molecule resulting from the joining of DNA segments This is often referred to as genetic engineering

The terminology "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) 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 , 1989, Molecular Cloning - A Laboratory Manual, 2nd Edition, CSH Laboratories, Ausubel et al , 1989, in Current Protocols in Molecular Biology, John Wiley & Sons Inc , N Y ) The size of the oligonucleotide will be dictated by the particular situation and ultimately on the particular use thereof and adapted accordingly by the person of ordinary skill An oligonucleotide can be synthesized chemically or derived by cloning according to well known methods For example, the skilled artisan will be able to adapt the length of the essentially homopolymeric stretch-containing oligo (the targeting stretch wherein the homopolymeric stretch has been modified in accordance with the teachings of the present invention), to particular needs, as a function of the targeted stretch and other parameters such as the sequence of the duplex, the conditions of the assay (and hence of the Tm) and the presence of additional sequences, flanking the essentially homopolymeric stretch (at the 5' and/or 3' end thereof)

The term "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 The term "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 α-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 deoxyπbonucleic 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 the present invention can be synthesized chemically or produced through recombinant DNA technology All these methods are well known in the art According to the present invention, the modified oligonucleotides are molecules comprising an essentially homopolymeric stretch or sequence composed of a single type of nucleotide and at least one type of modification which enables a destabilization of mismatches In a preferred embodiment, these modified oligonucleotides are a molecule composed of a single type of nucleotide (ribo- or deoxynbonucleotides, A, C, G, T or U) and containing at least one universal nucleoside As mentioned above, the length is between 10 and 50 nucleotides Of course, it should be recognized that in a case where more than one modification or nucleotide which destabilizes mismatches is used, it need not be the same type of "modification" In some embodiments of the present invention, the modified oligonucleotides of the present invention comprise an essentially homopolymeric stretch and a "3' lock" (see below) or a sequence enabling the creation of a restriction site As used herein, a "primer' defines an oligonucleotide which is capable of annealing to a target sequence, thereby creating a double stranded region or duplex which can serve as an initiation point for DNA synthesis under suitable conditions

"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 Examples of 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 In the case of 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 μg/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 at a temperature which is selected in view of the desired stringency room temperature (low stringency), 42°C (moderate stringency) or 65°C (high stringency) The selected temperature is based on the melting temperature (Tm) of the DNA hybrid Of course, RNA- DNA hybrids can also be formed and detected In such cases, the conditions of hybridization and washing can be adapted according to well known methods by the person of ordinary skill Stringent conditions will be preferably used (Sambrook et al ,1989, supra)

The types of detection methods in which 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 ³H, ¹⁴C, ³²P, and ³⁵S 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

As commonly known, 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 ³²P 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

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 Qβ 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) Preferably, amplification will be carried out using PCR Polymerase chain reaction (PCR) is carried out in accordance with known techniques See, e g , U S Pat Nos 4,683,195, 4,683,202, 4,800,159, and 4,965,188 (the disclosures of all three U S Patent are incorporated herein by reference) In general, PCR involves, a treatment of a nucleic acid sample (e g , in the presence of a heat stable DNA polymerase) under hybridizing conditions, with one oligonucleotide primer for each strand of the specific sequence to be detected An extension product of each primer which is synthesized is complementary to each of the two nucleic acid strands, with the primers sufficiently complementary to each strand of the specific sequence to hybridize therewith The extension product synthesized from each primer can also serve as a template for further synthesis of extension products using the same primers Following a sufficient number of rounds of synthesis of extension products, the sample is analysed to assess whether the sequence or sequences to be detected are present Detection of the amplified sequence may be carried out by visualization following EtBr staining of the DNA following gel electrophores, or using a detectable label in accordance with known techniques, and the like For a review on PCR techniques (see PCR Protocols, A Guide to Methods and Amplifications, Michael et al Eds, Acad Press, 1990)

Ligase chain reaction (LCR) 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) As used herein, 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 well known in the art

The term "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 Numerous types of vectors exist and are well known in the art

The term "allele" defines an alternative form of a gene which occupies a given locus on a chromosome

As commonly known, a "mutation" is a detectable change in the genetic material which can be transmitted to a daughter cell As well known, a mutation can be, for example, a detectable change in one or more deoxynbonucleotide For example, 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

As used herein, the term "purified" refers to a molecule having been separated from a cellular component Thus, for example, 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

The present invention also relates to a kit comprising the oligonucleotide primers of the present invention For example, 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 Of course, cDNA cloning kits could be adapted by inserting thereinto the primers of the present invention

BRIEF DESCRIPTION OF THE DRAWINGS

Having thus generally described the invention, reference will now be made to the accompanying drawings, showing by way of illustration a preferred embodiment thereof, and in which

Figure 1 (PRIOR ART) shows an example of the steps involved in generating cDNA libraries from mRNA Although a number of strategies can be used for cDNA library generation, of which only one is shown, all libraries require as a first step, a primer from which the reverse transcnptase (RT) can prime In the case of full-length cDNA libraries, an oligo d(T) primer is used because it anneals to the 3' poly (A) tail of the eukaryotic mRNAs In the case of prokaryotic, some viral, or other eukaryotic mRNAs which lack a poly (A) tail, a homopolymeric stretch of nucleoside 5'-monophosphates can be added to the 3' end of the mRNA For example, 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)) is then annealed to the mRNA and serves as primer for the RT

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 Although 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 The asterisks denotes mispainng resulting in destabilization of the duplex 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 (left) when hybridizing to oligo d(T)«Z The asterisks denote mispainng resulting in destabilization of the duplex and circles represent 3- nitropyrrole artificial mismatches

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 3¹ 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 ³H-test transcript Figure 3B shows the integrity of the in vitro generated transcript following fractionation on a formaldehyde 1 2% agarose gel, treatment with EN³HANCE, 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 α-³²P- dCTP The position of migration of truncated products are indicated by a filled circle and full length product by an arrow These results directly demonstrate correction of 3' mispnming by utilizing oligo d(T)*Z as primer during first strand synthesis

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

Oligo a, ⁵⁵⁶⁷GAAATTGACTCAGTACTATT⁵⁵⁸⁴,

O go b, ⁵⁴¹⁶GAAGGAAATGCTGTGGACC⁵⁵³⁵,

Ohgo c, ⁵¹⁹⁴TGTATAATAGAAAAGCAGAG⁵²¹³, O go d, ⁵⁰⁶⁸TTTTAAACAAGGACTCATAC⁵⁰⁸⁷, and

Ohgo e, ⁴⁷⁸¹AAGAGGAGTCTGAGGATAAC⁴⁸⁰⁰

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

Other objects, advantages and features of the present invention will become more apparent upon reading of the following non-restrictive description of preferred embodiments with reference to the accompanying drawing which is exemplary and should not be interpreted as limiting the scope of the present invention

DESCRIPTION OF THE PREFERRED EMBODIMENT

The demonstration of the destabilization effect of non-specific or artifactual duplex formation and of its concurrent effect on mismatch and/or mispnming events was carried out with an o go d(T) primer, modified with one or two universal analogues Whether such an introduction could result in increased discrimination between the perfectly matched target of that primer (i e the 3' poly (A) tail of the mRNA) and an imperfect matched sequenced (internal A-nch stretches) was analyzed

More specifically, an ohgo d(T) primer, called o go d(T)*Z was generated, in which two of the thymine bases were substituted by 3- nitropyrrole (Fig 2C) General teachings on 3-nιtropyrrole, the synthesis thereof and the like can be found for example in U S 5,438,131

To test whether this primer can reduce mispnming from internal A-nch sequences (Fig 2D), a cDNA clone from elF-4GII, a eukaryotic translation factor, was obtained When cDNA clones to this gene were initially isolated, only one of 5 clones had the correct 3' end Sequence characterization of these clones demonstrated that all the truncated clones were the result of internal priming by ohgo d(T) at four different sites (denoted as leftward arrows in Fig 3A) In vitro transcribed RNA generated from this clone thus serves as an excellent test reagent to determine the ability of the 3-nιtropyrrole substituted ohgo d(T) to decrease the number of mispnming events The quality of the in vitro transcribed RNA is shown in Fig 3B and demonstrates that the test template is intact This RNA was then annealed to ohgo d(T) or ohgo d(T)*Z, and reverse transcription performed with MMLV RT As shown in Fig 3C, use of o go d(T) on this template resulted in shorter than full-length products (>95%) generated as a result of internal priming (Fig 3C, lane 1) However, use of ohgo d(T)»Z as primer on the same template resulted in the majority (>95%) of products being full-length (Fig 3C, lane 2)

These results demonstrate that use of oligo d(T)*Z in reverse transcription reactions significantly improves the specificity for the 3' poly (A) tail and demonstrates the usefulness of this procedure in destabilizing non-specific duplex formation and more particularly for generating full length cDNAs

The sites of mispnming with ohgo d(T) on the control elF-4GII template were identified (Fig 4) This was done by fractionating the products of RT reactions performed with either ohgo d(T) or o go d(T)»Z on an alkaline agarose gel followed by transfer to a nylon membrane This membrane was then probed, by hybridization, with oligonucleotides designed to target various regions of the 3' untranslated region of elF-4Gll (oligonucleotides are labelled a, b, c, d and e in Fig 4A) As shown in Fig 4B, hybridization with oligonucleotide "a" detected correctly primed cDNA when both ohgo d(T) and oligo d(T)«Z were used as primer Hybridization with oligonucleotides b and c, detected a novel truncated product when the RT reaction was primed with ohgo d(T), indicating mispnming from an internal A-πch stretch with this primer (Fig 4B) Hybridization with oligonucleotides d and e, detected an additional novel, more abundant truncated product (denoted by arrowheads in Fig 4B) when the RT reaction was primed with ohgo d(T), indicating mispnming from a second internal A-nch stretch with this primer but not with oligo d(T)»Z (Fig 4B)

Mispnming event are common in Rapid Amplification of cDNA ends (RACE) 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-πch sequence It is expected that incorporation of at least one universal base (e g 3-nιtropyrrole) in the homopolymeric ohgo d(C) primer should significantly reduce such mispnming

The present invention is illustrated in further detail by the following non-limiting example

EXAMPLE 1 Destabilization of mispriming and reduction of mispriming using an oligo d(T)»l primer

To demonstrate that other "modifications" of nucleotides that destabilize hydrogen bonding between mismatched sequences could be used in accordance with the present invention, the o go d(T) primer was modified by inserting thereinto deoxynucleotide deoxyinosme (I)

An 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'deoxyιnosιne was incorporated into the oligonucleotide Reverse transcription reactions were performed on in vitro generated elF-4G mRNA templates (1 μg) with Superscript II™ (LifeTechnologies) under conditions recommended by LifeTechnologies Oligonucleotide primers that were utilized to prime the first strand synthesis were 0 1 μg of either Ohgo d(T)₁₅, o go d(T)»Z, or oligo d(T)»l The radioisotope α-³²P-dCTP (New England Nuclear) 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 phenol/chloroform, back extracted with an equal volume of water, passed through a G50™ spun column, and precipitated with 2M ammonium acetate and 2 5 volumes of ethanol The precipitate was washed with 70% ethanol, dried and resuspended in 20 μl of water An aliquot (5 ul) was loaded onto a 1 2% alkaline agarose gel and electrophoresis performed at 78 volts for 6 5 hours The gel was neutralized in 7% tπchloroacetic acid for 30 minutes, dried, and exposed to X-OMAT X-ray film (Kodak) at -70°C for 10 hrs with an intensifying screen

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)

Although the present invention has been described hereinabove by way of preferred embodiments thereof it can be modified, without departing from the spirit and nature of the subject invention as defined in the appended claims

Claims

WHAT IS CLAIMED IS:

1 A method for destabilizing non-specific duplex formation between an oligonucleotide and a target nucleic acid, comprising an incubation of said target nucleic acid with a modified oligonucleotide, wherein said modified oligonucleotide comprises a homopolymeric sequence having a modification which decreases or abrogates hydrogen bonding between said modified oligonucleotide and non-specific target sequences

2 The method of claim 1 wherein said modification is at least one universal base incorporated into said homopolymeric sequence

3 The method of claim 2 wherein said universal base is 3- nitropyrrole

4 The method of one of claims 1-3, wherein said oligonucleotide is a homopolymer comprising at least one nucleotide modification

5 A method for increasing the proportion of full length cDNA clones in a library, comprising a use of a modified ohgo d(T) during first strand synthesis, wherein said modified ohgo d(T) comprises a modification which decreases or abrogates hydrogen bonding between said modified ohgo d(T) and a non-specific target sequence, thereby increasing the proportion of full length cDNA clones

6 The method of claim 5, wherein said modification is at least one universal base incorporated into said o go d(T)

7 The method of claim 6, wherein said universal base is 3- nitropyrrole

8 The method of claim 5, wherein said modification is at least one chemically modified nucleoside incorporated into said ohgo d(T)

9 The method of claim 5, wherein said modification is at least one base analog incorporated into said ohgo d(T)

10 The method of claim 9, wherein said base analog is inosine

1 1 The method of claim 5, wherein said modification is at least one mismatch incorporated into said ohgo d(T)

12 The method of claim 5, wherein said modification is a phosphate or nbose modification incorporated into said ohgo d(T)

13 The method according to one of claims 5 to 12, wherein an enzyme capable of RNA-dependent DNA polymerization is used for said first strand synthesis

14 The method according to one of claims 5 to 12, wherein an enzyme capable of RNA-dependent RNA polymerization is used for said first strand synthesis

15 The method according to claim 14, wherein said enzyme is a reverse transcnptase selected from the group consisting of avian myoblastoid virus reverse transcnptase, munne moloney leukemia virus reverse transcnptase, and human immuno deficiency virus reverse transcnptase

16 A kit for the synthesis of cDNA, said kit comprising a modified ohgo d(T) primer, wherein said modified oligonucleotide includes a modification which decreases or abrogates hydrogen bonding between same and non-specific target sequences

17 A method for reducing mispnming events during DNA synthesis comprising a use of a modified oligonucleotide to prime said DNA synthesis, wherein said modified oligonucleotide comprises a homopolymeric sequence having a modification which decreases or abrogates hydrogen bonding between said modified oligonucleotide and a non-specific target sequence, thereby reducing mispnming events, while maintaining a formation of a duplex with a bona fide homopolymeric target sequence

18 The method of claim 17, wherein said modification is at least one universal base incorporated into said homopolymeric sequence

19 The method of claim 18, wherein said universal base is 3-nιtropyrrole

20 The method of claims 17, 18 or 19, wherein said oligonucleotide is a homopolymer

21. A method for reducing mispriming during 5' RACE comprising a use of a modified oligonucleotide to prime said 5' RACE, wherein said modified oligonucleotide comprises a homopolymeric sequence having a modification which decreases or abrogates hydrogen bonding between said modified oligonucleotide and a non-specific target sequence, thereby reducing mispriming events while maintaining a formation of a duplex with a bona fide homopolymeric target sequence.

22. The method of claim 21 , wherein said modification is at least one universal base incorporated into said homopolymeric sequence.

23. The method of claim 21 , wherein said universal base is 3-nitropyrrole.

24. The method of claim 21 , wherein said modification is at least one chemically modified nucleoside incorporated into said homopolymeric sequence.

25. The method of claim 21 , wherein said modification is at least one base analog incorporated into said homopolymeric sequence.

26. The method of claim 25, wherein said base analog is inosine.

27. The method of claim 21 , wherein said modification is at least one mismatch incorporated into said homopolymeric sequence.

28 The method of claim 21 , wherein said modification is a phosphate or nbose modification destabilizing mismatch recognition incorporated into said homopolymeric sequence

29 A kit for 5' RACE comprising a modified oligonucleotide primer, comprising a homopolymeric sequence having a modification which decreases or abrogates hydrogen bonding between said modified oligonucleotide and non-specific target sequences

30 A method for reducing mispnming during 3' RACE comprising a priming of said 3' RACE with a modified oligonucleotide, wherein said modified oligonucleotide comprises a homopolymenc sequence having a modification which decreases or abrogates hydrogen bonding between said modified oligonucleotide and a non-specific target sequence, thereby reducing mispriming events while maintaining a formation of a duplex with a bona fide homopolymenc target sequence

31 The method of claim 30, wherein said modification is at least one universal base incorporated into said homopolymenc sequence

32 A method for generating bona fide genetic markers comprising a use of a modified oligonucleotide to prime from homopolymenc stretches, wherein said modified oligonucleotide comprises a homopolymeric sequence having a modification which decreases or abrogates hydrogen bonding between said modified oligonucleotide and non-specific target sequences

33. The method of claim 32, wherein said modified oligonucleotide primes from an internal A-rich region in an Alu repeat.

34. A method for stabilizing duplex formation between an oligonucleotide and a target homopolymeric sequence comprising an incubation of said target homopolymeric sequence with a modified oligonucleotide, wherein said modified oligonucleotide comprises a homopolymeric tract having a modification which decreases or abrogates hydrogen bonding between same and non-specific target sequences.

35. A method for reducing mispriming during sequencing comprising a use of a modified oligonucleotide to prime DNA synthesis, wherein said modified oligonucleotide comprises a homopolymeric sequence having a modification which decreases or abrogates hydrogen bonding between same and non-specific target sequences.

36. A method to improve the discrimination between a binding of an oligonucleotide sequence to its targetted homopolymeric sequence versus a non-homopolymeric tract comprising an insertion into a homopolymeric tract of said oligonucleotide sequence of at least one modification which decreases or abrogates hydrogen bonding between same and said non- homopolymeric tract.

37. A method for increasing the proportion of full length cDNA clones in a library, comprising a use of a modified oligonucleotide during second strand synthesis from a 3' end-tailed first strand product, wherein said modified oligonucleotide comprises a homopolymeric sequence having a modification which decreases or abrogates hydrogen bonding between said modified oligo and a non-specific target sequence, thereby increasing the proportion of full length cDNA clones.