WO2007062495A1 - Selective terminal tagging of nucleic acids - Google Patents
Selective terminal tagging of nucleic acids Download PDFInfo
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- WO2007062495A1 WO2007062495A1 PCT/CA2005/001830 CA2005001830W WO2007062495A1 WO 2007062495 A1 WO2007062495 A1 WO 2007062495A1 CA 2005001830 W CA2005001830 W CA 2005001830W WO 2007062495 A1 WO2007062495 A1 WO 2007062495A1
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- sequence
- nucleic acid
- oligonucleotide
- template
- rna
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—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
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1096—Processes for the isolation, preparation or purification of DNA or RNA cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR
-
- 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
- C12Q1/6865—Promoter-based amplification, e.g. nucleic acid sequence amplification [NASBA], self-sustained sequence replication [3SR] or transcription-based amplification system [TAS]
Definitions
- This invention relates to a method for adding a terminal sequence tag to nucleic acid molecules and uses thereof for RNA transcription or DNA amplification, cloning or sequencing and identification of target nucleic acid molecules.
- Methods for determining mRNA sequences typically involve analyzing the DNA sequence of single clones of a cDNA library, which are derived by enzymatic production of double-stranded cDNA from the mRNA.
- Methods for determining the relative abundance of mRNA species typically involve quantifying the hybridization of a defined nucleic acid sequence to a complementary sequence in the mRNA population. Analysis of samples containing a relatively low quantity of mRNA generally involves amplification prior to the application of methods for determining the sequence or relative abundance of particular mRNA species. Amplification methods that proceed with linear kinetics during the course of the amplification reaction are less likely to introduce bias in the relative levels of different mRNAs than those that proceed with exponential kinetics (Shannon, U.S. Pat. No. 6,132,997).
- cDNA is synthesized from the mRNA by extension of the annealed primer-promoter using reverse transcriptase; the RNA strand of the resulting mRNAxDNA hybrid is partially hydrolyzed using RNase H; a second strand of DNA is synthesized from the cDNA by extension of the annealed mRNA fragments using DNA polymerase I (Gubler et al. (1983) Gene 25:263-269); and multiple copies of antisense RNA are synthesized from the second strand of DNA using an RNA polymerase.
- DNA polymerase I Gubler et al. (1983) Gene 25:263-269
- multiple copies of antisense RNA are synthesized from the second strand of DNA using an RNA polymerase.
- sequence tag For 5'-terminal mRNA sequences to be included in an amplified product, an arbitrary sequence, a "sequence tag", needs to be added to either the 5' ends of the mRNA or the 3' ends of the cDNA.
- This sequence tag provides a terminal priming site needed for amplification of the cDNA that was synthesized from the initial priming site, typically the 3'-terminal poly(A) of mRNA.
- Three general methods for providing a terminal priming site on mRNA or cDNA for the purposes of nucleic acid amplification are described below.
- cDNA having a 3'-terminal arbitrary sequence is synthesized from the ligated mRNA products by extension of an annealed oligo(dT) primer using reverse transcriptase. Since this process requires the performance of two hydrolytic steps on the mRNA, any contaminating hydrolytic activities in the enzymes and the alkaline reaction conditions can cause the loss of intact mRNA. In addition, T4 RNA ligase is less efficient with longer nucleic acid substrates.
- cDNA is synthesized from mRNA by extension of an annealed primer having a 3'-terminal oligo(dT) linked to a 41 -nt arbitrary sequence using reverse transcriptase.
- an oligodeoxyribonucleotide having a 44-nt arbitrary sequence, a 5'-terminal phosphate and a blocked 3' end, is added to the 3' ends of the cDNA using T4 RNA ligase.
- the ligated cDNA products are amplified using PCR with primers derived from the 5'-terminal half of each arbitrary sequence.
- the resulting amplified products are purified and amplified using a second PCR this time with nested primers derived from the 3'-terminal half of each arbitrary sequence.
- a process for the synthesis and cloning of cDNA corresponding to the 5' ends of mRNA using a template- v switching oligonucleotide that hybridizes to the 5'-terminal CAP of mRNA.
- the method comprises contacting RNA with a cDNA synthesis primer which can anneal to RNA, a suitable enzyme which possesses reverse transcriptase activity, and a template switching oligonucleotide under conditions sufficient to permit the template-dependent extension of the primer to generate an mRNA:cDNA hybrid.
- the template switching oligonucleotide hybridizes to the CAP site at the 5' end of the RNA molecule and serves as a short, extended template for CAP-dependent extension of the 3'-end of the single stranded cDNA that is complementary to the template switching oligonucleotide.
- the resulting full-length single stranded cDNA includes the complete 5'-end of the RNA molecule as well as the sequence complementary to the template switching oligonucleotide, which can then serve as a universal priming site in subsequent amplification of the cDNA.
- the template switching oligonucleotide hybridizes to the CAP site at the 5' end of the mRNA and forms basepair(s) with at least one nucleotide at the 3' end of the cDNA of an mRNA-cDNA intermediate. Since this process is based upon the specific interaction with the CAP of an mRNA and the 3' end of a cDNA in an mRNA-cDNA intermediate, it is unlikely to be applicable for adding terminal sequence tags to nucleic acid molecules that are single-stranded or are without a CAP structure.
- the present invention seeks to meet these needs and other needs.
- the present invention provides methods, kits and reagents for adding at least one terminal nucleic acid sequence (a sequence tag) to target nucleic acid molecules.
- the present invention provides in a first aspect thereof, a first oligonucleotide which may comprise sequentially (in a 5'-> 3' direction), an overhanging portion and an hybridizing portion.
- the present invention more particularly relates to a first oligonucleotide which may comprise; i) an overhanging portion which may comprise, for example, a first sequence tag and; ii) an hybridizing portion which may be able to hybridize to at least one target nucleic acid molecule.
- the overhanging portion may be substantially non-hybridizable to a target nucleic acid molecule.
- the overhanging portion may be substantially non-hybridized to a target nucleic acid molecule upon hybridization of the hybridizing portion with the target nucleic acid molecule.
- the overhanging portion may comprise, for example, one or more sequence tags.
- the overhanging portion may be able to serve as a template for a polymerase, such as for example, a DNA polymerase.
- the first sequence tag may comprise a sequence which is defined in accordance with the need of the user (a user-defined sequence), and although exemplary first sequence tags are given herein, it is to be understood that the choice of the first sequence tag is not intended to be limited.
- the hybridizing portion may comprise, for example, a nucleic acid sequence selected from the group consisting of 1) a random sequence and 2) a nucleic acid sequence substantially complementary (e.g., 80 to 100% complementarity over the entire sequence or portion of sequences) to a portion located at a 3'-end of a target nucleic acid molecule (with respect to the 5'-> 3' direction).
- a nucleic acid sequence selected from the group consisting of 1) a random sequence and 2) a nucleic acid sequence substantially complementary (e.g., 80 to 100% complementarity over the entire sequence or portion of sequences) to a portion located at a 3'-end of a target nucleic acid molecule (with respect to the 5'-> 3' direction).
- the sequence tag may be located near the 5'-end of the first oligonucleotide and the hybridizing portion may be located near the 3'-end of the first oligonucleotide.
- the hybridizing portion may be able, more particularly to hybridize to the 3'-end of a target nucleic acid molecule in such a manner that the overhanging portion extends past the 3'-end of the target.
- the first oligonucleotide may also comprise a blocked 3'-end (3 1 - terminus).
- the blocked 3'-end may prevent, for example, the first oligonucleotide from functioning as a primer for primer extension using the first templates as template.
- the present invention also relates to a plurality of first oligonucleotides each of which may comprise; i) an overhanging portion which may comprise a first sequence tag and; ii) an hybridizing portion which may be able to hybridize to at least one target nucleic acid molecule.
- the plurality of first oligonucleotides may each comprise a blocked 3'-end.
- the hybridizing portion of each of first oligonucleotides may comprise, for example, a random sequence.
- the random sequence of each of the first oligonucleotides may be, for example, substantially different from one another (in terms of nucleic acid composition and/or length, etc.).
- each of first oligonucleotides may comprise, for example, a nucleic acid sequence substantially complementary (e.g., 80 to 100% complementarity over the entire sequence or portion of sequences) to a portion located at a 3'-end of a target nucleic acid molecule.
- the first sequence tag of each of the first oligonucleotides may be identical or substantially identical (e.g., 80 to 100% sequence identity) to one another or to a portion thereof. In some circumstances, it may be useful that the first sequence tag comprises a sequence complementary to a desired sequence.
- the first sequence tag may comprise, for example, a promoter sequence.
- the first sequence tag may be a promoter sequence.
- the promoter sequence may be selected, for example, from the group consisting of a RNA polymerase promoter sequence, a DNA polymerase promoter sequence etc.
- RNA polymerase promoter sequence may be selected, for example and without limitation, from the group consisting of bacteriophage RNA polymerases promoters such as, the bacteriophage T7 RNA polymerase, the phage T3 RNA polymerase, the Salmonella phage sp6 RNA polymerase etc.
- the first oligonucleotide may comprise, for example, a promoter and initiation sequences which may be specific for a desired RNA polymerase. It will occur to those of skill in the art that other suitable promoter and initiation sequences may be used to achieve desirable levels of transcription of RNA as described herein.
- the hybridizing portion is preferably not a oligo(dT) sequence or if a oligo(dT) is used, the 3'-end may preferably be blocked.
- the first sequence tag is not intended to be limitative and may be an arbitrary sequence having any combination of purines and pyrimidines, including but not limited to G, A, T or C (natural or modified) arranged to form a sequence of any desired length.
- the sequence tag may be defined by the user to have a specific length and base composition to provide a template for accurate extension of the nucleic acid molecules.
- the sequence tag may be deoxy- and/or ribonucleotides as long as it provides a template for the enzymatic extension of the nucleic acid molecules.
- the sequence tag may, for example, be substantially free of symmetry elements, such as direct and inverse repeats, and it may provide a template for extension of the nucleic molecules in forming a 3'-terminal sequence tag.
- the complementary sequence of the first sequence tag may provide a suitable sequence that may be used as a site for hybridizing and extending an oligonucleotide primer or for hybridizing an oligonucleotide template, which may be used for extension or detection of the tagged nucleic acid molecules or for other purposes.
- sequence tag portion of the oligonucleotide may comprise a sequence defined by the user to carry out the different steps of a method of the present invention, however any suitable sequence tag may be used to carry out the method of the present invention and this portion of the oligonucleotide is not intended to be limited to a specific nucleotide sequence.
- the sequence tag may provide the nucleic acid molecule with a defined sequence at its terminus and this sequence tag may subsequently serve as an hybridization site (a means for hybridizing) for 1) subsequent amplification of the nucleic acid molecule with a primer that comprises a nucleic acid sequence complementary to the sequence tag, 2) subsequent add a further desired sequence to the nucleic acid molecule by using the methods described herein, etc.
- the first sequence tag may comprise a functional sequence such as a RNA polymerase promoter sequence and therefore may directly be used to amplify RNA from the tagged nucleic acid target.
- the random sequence portion of the first oligonucleotide may be any number of nucleotides in length such as between about 4 and about 9 (or from about 4 to 15).
- the random sequence may comprise an equal representation of G, A, T and C at each of the different positions.
- Wobble bases such as inosine (I) may also be used instead of the standard bases at any of the positions.
- nucleotides contained in the random sequence may be chemically modified for example, 2'-0 methylated nucleotides, phosphorothioates or any such chemical modifications that render the nucleotide(s) inert to nucleases.
- the first oligonucleotide may comprise a blocked 3'-end.
- the 3' terminus of the oligonucleotides may be chemically blocked with, for example, C3 propyl spacer, amine group (NH 2 ), phosphate or any other chemical modifications that render the oligonucleotide mixture inert as a primer for primer extension using either a DNA- or RNA-directed DNA polymerase.
- the present invention also provides a plurality of first oligonucleotides each first oligonucleotides may comprise 1 ) one or more first sequence tag and 2) hybridizing portion selected from the group consisting of a random sequence and a nucleic acid sequence substantially complementary to a portion (of a target) located at a 3'-end of the target nucleic acid molecule (with respect to the 5'-> 3' direction) and combination thereof.
- the hybridizing portion of each of the first oligonucleotides may be the same or different.
- sequence tags a plurality of user defined sequence tags
- the oligonucleotide used in the present method may comprise a specific hybridizing portion able which is substantially complementary to a portion (of a target) located at a 3'-end of the target nucleic acid molecule and a plurality of user defined sequence tags.
- Each of the nucleic acid sequence substantially complementary to a portion of a target nucleic acid molecule located at a 3-'end of the target may be the same or different from one another.
- the first oligonucleotide may be composed of nucleic acid sequence defined by the user (e.g., a specific nucleic acid sequence may be used to carry the methods of the present invention).
- the hybridizing portion may be defined to be complementary to a corresponding portion of a known nucleic acid target sequence. Therefore, a portion of the known sequence of a target nucleic acid which is located at the 3'-end (e.g., extreme 3'-end (or terminal sequence)) of the target may be used to design a complementary hybridizing portion.
- a suitable hybridizing portion of the first oligonucleotides may also be composed of a random sequence.
- a mixture of first oligonucleotides comprising a library of random sequences attached to the first sequence tag may be used.
- the sequence of the first oligonucleotide used for terminal tagging may preferably comprise random sequences.
- the target nucleic acids may encompass unique species or multiple species.
- the first oligonucleotides used to add a terminal tag to unique or multiple species the same to those described above. It is to be understood herein that when a terminal tag is to be added to multiple species contained within a sample (solution, tissue, etc.) a plurality of first oligonucleotides comprising: 1) a first sequence tag and 2) a random sequence, may be used. Again in order to increase the chance to add a tag to several unrelated species (multiple species) of target nucleic acid molecules, the random sequence of each of the first oligonucleotides may preferably be different.
- each first oligonucleotides may comprise; 1 ) an identical sequence tag and 2) a different random sequence.
- the first oligonucleotides may further comprise a blocked 3'-end.
- the present invention also relates to the addition of a second sequence tag to a first template.
- the second sequence tag may be added, for example, to a first template comprising a first sequence tag.
- the present invention relates in a further aspect thereof, to a second oligonucleotide which may comprise; i) an overhanging portion which may comprise a second sequence tag (a desired sequence or a sequence of interest); and ii) an hybridizing portion which may comprise a first sequence tag.
- the overhanging portion may be substantially non-hybridizable to a target nucleic acid molecule or first template.
- the overhanging portion may be substantially non-hybridized to a target nucleic acid molecule or first template upon hybridization of the hybridizing portion with the target nucleic acid molecule or first template.
- the overhanging portion may serve as a template for a polymerase.
- the second oligonucleotide may comprise sequentially (in a 5'-> 3' direction); a) a 5'- overhanging portion which may comprise a second sequence tag (a desired sequence or sequence of interest) and; b) an hybridizing portion which may comprise a first sequence tag.
- the second oligonucleotide may further comprise a blocked 3' (a blocked 3'-terminus).
- the blocked 3' terminus may prevent, for example, the second oligonucleotide from functioning as a primer for primer extension using the first templates as template.
- the hybridizing portion may be at the 3'-end of the oligonucleotide. Further in accordance with the present invention, the overhanging portion may be located at the 5'-end of the second oligonucleotide and the hybridizing portion may be located at the 3'-end of the second oligonucleotide. For example, the overhanging portion may be 5' relative to the hybridizing portion.
- first sequence tag of the second oligonucleotide may be identical or substantially identical to the first sequence tag of the first oligonucleotide or to portions thereof. It is therefore, to be understood herein that the first sequence tag may be substantially complementary to the complementary first sequence tag or portions thereof of the first template.
- the second sequence tag (desired sequence or sequence of interest) may be, for example, selected from the group consisting of a promoter sequence, a restriction site, or any other sequence of choice and combination of several sequences of choice.
- the second sequence tag may comprise, more particularly, a promoter sequence.
- the promoter sequence may comprise for example, a RNA polymerase promoter sequence, a DNA polymerase promoter sequence etc.
- RNA polymerase promoter sequence may be selected, for example and without limitation, from the group consisting of bacteriophage RNA polymerases promoters such as, a T7 RNA polymerase promoter sequence, a sp6 RNA polymerase promoter sequence, etc.
- the second oligonucleotide may comprise a promoter and initiation sequences which may be specific for a desired RNA polymerase such as the bacteriophage T7 RNA polymerase, the phage T3 RNA polymerase, the Salmonella phage sp6 RNA polymerase, etc. It will occur to those of skill in the art that other suitable promoter and initiation sequences may be used to achieve desirable levels of transcription of RNA as described herein.
- the second sequence tag may be of a particular length and base composition to allow specific and efficient annealing to the (complementary first) sequence tag of the first template under conditions, including those of an enzymatic DNA polymerization reaction.
- the second oligonucleotide may thus comprise, for example, in its overhanging portion, a sequence of interest such as the plus (+) sense sequence of a promoter and its transcription initiation site.
- the promoter template may be of a particular length and base composition to allow specific and desirable synthesis of double-stranded promoters by extension of the first template under the conditions of an enzymatic DNA polymerization reaction.
- the resulting double-stranded promoter may contain sufficient information to allow specific and desirable (operative) binding of a RNA polymerase and initiation of transcription at the desired site.
- the second sequence tag may be a sequence allowing for its (operative) recognition and cleavage by a restriction endonuclease site.
- the restriction endonuclease site may be located, for example, at a 5- terminus (5'-end) or may be embedded within the second oligonucleotides.
- the first sequence tag comprises a sequence complementary to a desired sequence.
- the overhanging portion (5'-overhanging portion) and the hybridizing portion of oligonucleotides of the present invention may be covalently attached to each other. More particularly, the overhanging portion (5'-overhanging potion) and the hybridizing portion may be made of consecutive nucleic acid separated or not by other nucleic acids or other type of spacers.
- the oligonucleotides of the present invention may also have a 3'-terminal sequence that reduces annealing to itself or another primer in the reaction such that a primer would be extended using itself or another primer as template in a DNA or RNA amplification reaction, hence producing what is described in the art as "primer-dimers”.
- the target nucleic molecules may be any type of nucleic acid having an end (3'-end) extendable by a polymerase.
- the target nucleic acid molecule may also have a portion substantially complementary to a first or second oligonucleotide.
- the target nucleic acid may be composed of natural nucleic acids or modified nucleic acids.
- the target nucleic acid molecule may be for example, a RNA molecule, a DNA molecule, a RNA/DNA hybrid, etc.
- DNA molecule may be used to carry out the present invention, such as for example and without limitation, a single-stranded DNA molecule, a double-stranded DNA molecule, a partially double-(and single) stranded DNA molecule, a DNA/RNA hybrid, DNA library etc.
- the method of the present invention may comprise a step of transforming the double- stranded DNA molecule into a substantially single-stranded DNA molecule.
- Double- stranded DNA may be made single-stranded by using, for example, chemical, enzymatic, mechanical or thermal methods. It is also to be understood herein that DNA molecules may originate from various sources, including without limitation, mammalian genomic DNA (human, animal, etc.), cDNA, bacterial DNA, viral DNA, insect DNA, etc.
- a target RNA molecule may be any ribonucleic acid molecule or library of ribonucleic acid molecules containing a 3'-OH group.
- the RNA molecules may be for example and without limitation, a messenger RNA (mRNA), a heterogeneous nuclear RNA (hnRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), bacterial RNA, viral RNA, single-stranded RNA, double-stranded RNA, antisense-RNA etc. Double-stranded RNA may be made single-stranded by using chemical, enzymatic, mechanical or thermal methods.
- the RNA molecule is a mRNA.
- the DNA molecule is a complementary DNA (cDNA).
- An initial mRNA target may be transformed in a cDNA target by using standard methods of reverse transcription known in the art.
- cDNA molecules may be formed by contacting a mixture containing mRNA with a primer comprising a terminal sequence substantially complementary to the mRNA, under conditions such that, the terminal sequence of the primer anneals with the mRNA and is extended using the mRNA as template.
- This method is commonly effected and may be performed for example, by using a oligo(dT) primer which hybridizes to the poly-A tail found at the 3'-end of eukaryotic mRNA and an enzyme which may suitably use a mRNA as template to generate a complementary DNA molecule.
- a suitable enzyme having these characteristic is, for example, a Reverse transcriptase enzyme.
- a primer complementary to the 3'-end of the known target mRNA may be used to generate a cDNA.
- the cDNA may be prepared from total RNA or purified mRNA containing a single or multiple species, using an oligo(dT) as primer and reverse transcriptase for extending the primer.
- the RNA may be removed from the cDNA by using chemical, enzymatic, mechanical or thermal methods.
- non polyA- containing RNA may be transformed into a cDNA by using an oligonucleotide which hybridizes to a known sequence substantially near a 3'-end.
- the present invention also relates to methods for adding a sequence tag to target nucleic acid molecules.
- a terminal tag may be added to the 3'-end of a target nucleic acid molecule.
- the present invention provides in one aspect thereof, a method for adding a sequence tag (a terminal sequence tag) to a target nucleic acid molecule, the method may comprise, for example, contacting the target nucleic acid molecule with at least one oligonucleotide which may comprise; i) a 5'-overhanging portion comprising a sequence tag, and; ii) an hybridizing portion which may be selected from the group consisting of a) a random sequence, and; b) a sequence which may be substantially complementary to a portion (of a target nucleic acid molecule) located, for example, at a 3'-end of the target nucleic acid molecule, under conditions which may allow hybridization (annealing) of the hybridizing portion with the target nucleic acid molecule.
- the present invention relates to a method for adding a sequence tag to a target nucleic acid molecule, the method may comprise; contacting the target nucleic acid molecule with at least one oligonucleotide which may comprise; i) a 5'- overhanging portion comprising a sequence tag, and; ii) an hybridizing portion comprising a random sequence, under conditions allowing hybridization (annealing) of the hybridizing portion with the target nucleic acid molecule.
- the present invention relates to a method for adding a sequence tag to a target nucleic acid molecule which may comprise; contacting the target nucleic acid molecule with at least one oligonucleotide which may comprise; i) a 5'-overhanging portion comprising the sequence tag, and; ii) an hybridizing portion comprising a sequence substantially complementary to a portion located at a 3'-end of the of a target nucleic acid molecule, under conditions which may allow hybridization (annealing) of the hybridizing portion with the target nucleic acid molecule.
- the method may further comprise a step of: extending the target nucleic acid molecule and at least one oligonucleotide.
- the method may, for example, further comprise a step of; extending the target nucleic acid molecule whereby the oligonucleotide may remain unextended.
- at least one oligonucleotide may further comprise a blocked 3'-end.
- the method may further comprise a step of: extending the target nucleic acid molecule to generate a first template comprising a complementary first sequence tag whereby the oligonucleotide may remain unextented.
- the present invention also particularly relates to a method wherein a plurality of target nucleic acid molecules may each be tagged, the method may comprise the step of contacting the plurality of target nucleic acid molecules with a plurality of oligonucleotides each comprising; i) a 5'- overhanging portion comprising a sequence tag, and; ii) an hybridizing portion selected from the group consisting of a) a random sequence and b) a sequence substantially complementary to a portion of a target nucleic acid molecule located at a 3'-end of the target, under conditions which may allow hybridization of the hybridizing portion (a second portion) with the target nucleic acid molecules.
- the present invention relates to a method wherein a plurality of target nucleic acid molecules may each be tagged, the method may comprise the step of contacting the plurality of target nucleic acid molecules with a plurality of oligonucleotides each comprising; i) a 5'-overhanging portion comprising a sequence tag, and; ii) an hybridizing portion comprising a random sequence, under conditions which may allow hybridization of the hybridizing portion with the target nucleic acid molecules.
- the method may comprise a step of extending the plurality of target nucleic acid molecules.
- the present invention provides in a further aspect thereof, a method for adding a terminal sequence tag to a target nucleic acid molecule which may comprise the steps of, for example; a. contacting the nucleic acid molecule with an oligonucleotide which may comprise (include), for example; i. a 5'-overhanging portion which may include a first sequence tag; ii. an hybridizing portion which may be able to hybridize to the target nucleic acid molecule, and; iii. a blocked 3'-end, the contacting step may be effected under conditions which may allow hybridization (annealing) of the hybridizing portion with the target nucleic acid molecule and; b. extending the target nucleic acid molecule to generate a first template comprising a complementary first sequence tag.
- an oligonucleotide which may comprise (include), for example; i. a 5'-overhanging portion which may include a first sequence tag; ii. an hybridizing portion which may be
- the present invention provides in an additional aspect thereof, a method for adding a terminal sequence tag to a plurality of target nucleic acid molecules, the method may comprise the steps of, for example a. contacting the plurality of nucleic acid molecules with a plurality of oligonucleotides each of which may comprise, for example; i. a 5'-overhanging portion which may comprise a first sequence tag; ii. an hybridizing portion which may be able to hybridize to a target nucleic acid molecule and; iii. a blocked 3'-end, wherein the contacting step may be effected under conditions which may allow hybridization of the hybridizing portion with the target nucleic acid molecule and; b. extending the plurality of target nucleic acid molecules to generate a plurality of first templates each comprising a complementary first sequence tag.
- the method may further comprise the step of carrying extension of the target nucleic acid molecule by providing the mixture of target and first oligonucleotide with conditions and reagents allowing extension.
- the extension step may be performed by a polymerase as described herein.
- oligonucleotides used in the present methods are as described herein.
- a first oligonucleotides may be used in the exemplary embodiments of the method of the present invention.
- the hybridizing portion of the oligonucleotide may be able to hybridize at the 3'-end of the target nucleic acid molecule, for example at the extreme 3'-end.
- the S'-overhanging portion of the oligonucleotide may serve as a template for a ribonucleotide or deoxyribonucleotide polymerization reaction.
- the hybridizing portion of each oligonucleotide of the plurality of oligonucleotides may be substantially different from one another (in terms of nucleic acid composition and/or length, etc.).
- the first sequence tag of each oligonucleotide may be identical or substantially identical to one another (e.g., from about 80 to 100% sequence identity, from about 90 to 100% sequence identity, from about 95 to 100% sequence identity).
- the first sequence tag of each oligonucleotide may be substantially different from one another (in terms of nucleic acid composition and/or length, etc.).
- a random sequence refers to a sequence selected amongst a population of (random) sequences which have been isolated or synthesized in such a manner that each of the four bases (modified or not) are represented at every position in the population.
- the identity of the selected "random sequence” may be known.
- the definition of "random sequence” encompasses a sequence made by randomization of nucleotides.
- the oligonucleotide may be made, for example, from deoxyribonucleic acid (deoxyribonucleotides).
- the target nucleic acid molecule may comprise, for example DNA, such as a single-stranded DNA molecule.
- the single-stranded DNA molecule may be a positive strand DNA molecule or a negative strand DNA molecule.
- the negative strand DNA molecule may be, for example, a complementary DNA (cDNA).
- the target nucleic acid molecule may comprise, for example, RNA, such as for example, a messenger RNA or a portion thereof or an antisense RNA or portion thereof.
- the target nucleic acid(s) may comprise an unknown sequence or a known sequence.
- the first oligonucleotides used to carry out methods of the present invention may be defined to be complementary to a desired known sequence of the target.
- the target nucleic acid(s) comprises an unknown sequence
- the first oligonucleotides used to carry out the method of the present invention may be a plurality of first oligonucleotides which may each comprise a random sequence.
- At least one random sequence of at least one oligonucleotide may hybridize to the target in such a way that the 5'-overhanging portion (sequence tag) may be used as a template for a DNA polymerase and the 3'- end of the target may be used as a primer extension site for the DNA polymerase.
- a first or second oligonucleotide may hybridize to the 3'-end of a target nucleic acid (or first template) (through the hybridizing portion of the oligonucleotide) in such a manner that the 5'-overhanging portion remains non-hybridized and extends past the extreme 3'-end of the target, the DNA polymerase enzyme may use the 5'- overhanging portion (comprising a sequence tag) as a template and the 3'-end of the target as a primer extension site. When the 3'-end of the oligonucleotide is blocked, the oligonucleotide may not serve as a site for primer extension by the DNA polymerase.
- a terminal sequence tag may be added to an unknown target nucleic acid molecule (e.g., single species or multiple species) by contacting a sample containing the unknown target nucleic acid with a mixture of first oligonucleotides (i.e., a library of first oligonucleotides) each comprising a first sequence tag and a random sequence under conditions allowing hybridization of the target and first oligonucleotides and extending the target using the first sequence tag portion of the first oligonucleotides as template.
- a mixture of first oligonucleotides i.e., a library of first oligonucleotides
- Each of the random sequences comprised within the first oligonucleotides may be the same or different (the plurality of first oligonucleotides may thus represent a library of random sequences attached to a first sequence tag of invariable or low variability (e.g., 0 to 20% sequence variation amongst first tag sequence of each of the first oligonucleotides).
- the terminal tagging of unique species of target nucleic acid having a known nucleic acid sequence may thus be made by contacting a sample (e.g., a solution, tissue, etc.) containing the target nucleic acid with a first oligonucleotide which may include a first portion comprising a first tag sequence and a second portion selected from the group consisting of a random sequence or a sequence substantially complementary to a corresponding portion located at a 3'-end of the target nucleic acid molecule under conditions allowing hybridization of the target with the first oligonucleotide and extending the target using the sequence tag portion of the first oligonucleotide as template.
- a sample e.g., a solution, tissue, etc.
- a first oligonucleotide which may include a first portion comprising a first tag sequence and a second portion selected from the group consisting of a random sequence or a sequence substantially complementary to a corresponding portion located at a 3'-end of
- the terminal tagging of unique species of a target nucleic acid having an unknown nucleic acid sequence may be made by contacting a sample (e.g., a solution, tissue, etc.) containing the target nucleic acid with a first oligonucleotide which may include a first portion comprising a first tag sequence and a second portion comprising a random sequence under conditions allowing hybridization of the target and first oligonucleotides and extending the target using the sequence tag portion of the first oligonucleotides as template.
- a mixture of first oligonucleotides comprising a library of random sequences attached to the first tag sequence may be used.
- the terminal tagging of multiple target nucleic acid species having known nucleic acid sequence may be made by contacting a sample (e.g., a solution, tissue, etc.) containing the multiple target nucleic acid species with a mixture of first oligonucleotides each of which may include a first portion comprising a first tag sequence and a second portion selected from the group consisting of a random sequence or a sequence substantially complementary to a corresponding portion located at a 3'end of said target nucleic acid molecule.under conditions allowing hybridization of the target with the first oligonucleotides and extending the target using the sequence tag portion of the first oligonucleotides as template.
- a sample e.g., a solution, tissue, etc.
- first oligonucleotides each of which may include a first portion comprising a first tag sequence and a second portion selected from the group consisting of a random sequence or a sequence substantially complementary to a corresponding portion located at a 3'end
- the second portion of the first oligonucleotide may be different for each oligonucleotide whereas the first portion may be substantially identical for each oligonucleotide.
- the terminal tagging of multiple nucleic acid species having an unknown nucleic acid sequence may be made by contacting a sample (e.g., a solution, tissue, etc.) containing the nucleic acid with a mixture of first oligonucleotides each of which may include a first portion comprising a first tag sequence and a second portion comprising a random sequence under conditions allowing hybridization of the target with the first oligonucleotides and extending the target using the sequence tag portion of the first oligonucleotides as template.
- a sample e.g., a solution, tissue, etc.
- first oligonucleotides each of which may include a first portion comprising a first tag sequence and a second portion comprising a random sequence under conditions allowing hybridization of the target with the first oligonucleotides and extending the target using the sequence tag portion of the first oligonucleotides as template.
- sequence tag may be added to the terminal end of nucleic acid molecules by repeating one or more steps of the method of the present invention.
- the first sequence tag may comprise a nucleic acid sequence which may be selected, for example, from the group consisting of a promoter sequence, an endonuclease restriction site, an hybridization site and combination thereof and/or any sequence of interest.
- the oligonucleotides and target nucleic acid molecules may be allowed to anneal by heating a mixture of these two components at an elevated temperature (e.g., greater than about 37°C) for a period of time and then incubating at a temperature that is desirable for enzymatic extension of the nucleic acid molecules, for example by a DNA Polymerase.
- the desirable temperature and other enzymatic conditions are determined based on the characteristics of the enzyme used for the reaction and guidance for such conditions is provided by the manufacturer or is known in the art.
- the target nucleic acid molecules may therefore, be extended by using a DNA polymerase, which may be any enzyme capable of synthesizing DNA by extending a DNA or RNA primer using a DNA or RNA template.
- the DNA polymerase may be (substantially) free of exonuclease activities, either 3' to 5' or 5' to 3' although not necessarily. Preparations containing the DNA polymerase may be substantially free of agents capable of nucleic acid hydrolysis. Examples of DNA polymerase which may be used include, without limitation, [Klenow exo ' DNA polymerase, Bst DNA polymerase, AMV and M-MLV reverse transcriptases etc.
- the DNA polymerase reaction therefore may comprise the desirable concentrations of cofactors and deoxynucleoside triphosphates for DNA synthesis using the particular DNA polymerase and may be performed under the conditions of pH, ionic strength and temperature that are desirable for the enzyme that is used. Such reaction conditions are known to those skilled in the art.
- the reaction is performed for a sufficient period of time to allow extension of the nucleic acid molecules using the oligonucleotides as template.
- the reaction may be terminated using any chemical, enzymatic, mechanical or thermal methods, and the extended nucleic acid molecules may be purified from the unused oligonucleotides using size exclusion or any other suitable separation method known in the art.
- the resulting nucleic acid molecules have a terminal sequence tag that is complementary to the sequence tag contained in the oligonucleotide
- the present invention also relates to the addition of a second sequence tag to a target nucleic acid or to a first template.
- the second sequence tag may comprise any sequence of interest as well as multiple sequence of interest.
- Exemplary sequence of interest may include, for example, a promoter, an endonuclease restriction site, etc.
- the present invention thus also relates to a method for adding a second sequence tag to a target nucleic acid or to a first template comprising a complementary first sequence tag described herein, the method may comprise the steps of; i) contacting the first template with a second oligonucleotide which may comprise a) a 5'-overhanging portion which may include the second sequence tag (a sequence of interest); and b) an hybridizing portion which may comprise a first sequence tag or a portion of a first sequence tag and substantially identical sequences (80 to 100% identity with first sequence tag or fragments thereof) the contacting step may be effected, for example under conditions allowing hybridization of the hybridizing portion with the first template and; ' ii) extending wholly or partially the first template and second oligonucleotide to generate a second template.
- the present invention also relates to the addition of a second sequence tag to a plurality of target nucleic acids or to a plurality of first templates comprising a complementary first sequence tag.
- the present invention also relates to a method which may comprise the steps of; i) contacting the plurality of first templates with a plurality of second oligonucleotides each of the second oligonucleotides may comprise; a) a 5'-overhanging portion which may include the second sequence tag (a sequence of interest); and b) an hybridizing portion which may comprise a first sequence tag or a portion of a first sequence tag or substantially identical sequences (80 to 100% identity with first sequence tag or portion thereof) under conditions which may allow hybridization of the hybridizing portion with the plurality of first templates and; ii) extending wholly or partially the plurality of first templates and second oligonucleotides to generate a second template.
- the first sequence tag comprised in the first oligonucleotide may be identical or substantially identical (e.g., 80-100% sequence identity over the entire sequence or over a portion of the sequence) to the first sequence tag or a portion of a first sequence tag of the second oligonucleotide.
- the second oligonucleotide may thus anneal to the totality or to portion of complementary first sequence tag.
- the first sequence tag of each second oligonucleotide may alternatively be identical to one another.
- the 5'-overhanging portion of the second and first oligonucleotide may be different from one another of alternatively may be substantially identical (80-100% sequence identity) to one another.
- the hybridizing portion may be able to hybridize at the 3'-end (extreme 3'-end) of the first template and the 5'-overhanging portion may serve as a template for a ribonucleotide or deoxyribonucleotide polymerization reaction.
- the 3'-end of the first template may serve as a primer extension site for a DNA polymerase.
- the firs, second or both oligonucleotides may be made of ribo- or deoxyribonucleic acid or combination thereof.
- the first template and second template may comprise DNA or RNA.
- the second template may be at least partially double-stranded or totally double-stranded.
- the second template may be double-stranded in the promoter region and the remaining may be single-stranded.
- the second template may comprise a double-stranded second sequence tag region such as for example, a double-stranded promoter region, a double-stranded restriction endonuclease region etc.
- the double-stranded promoter region may comprise, for example, a double-stranded RNA polymerase promoter.
- the double-stranded promoter may be located at a 5'-end or 3'-end of a first or second template.
- the second sequence tag (sequence of interest) may be selected from the group consisting of a promoter sequence, an endonuclease restriction site, an hybridization site and combination thereof and/or any other sequence of interest.
- a RNA polymerase promoter sequence may be added to a target DNA molecule by forming, for example, a first DNA template as described herein and forming a second DNA template having a double-stranded RNA polymerase promoter sequence (see schematic of Figure 4 for illustration). The remaining of the second DNA template may be double-stranded or not.
- the first, second or both oligonucleotides may be composed in part of nucleotides other than deoxyribonucleotides provided that they may still function as template for DNA polymerization.
- the reaction may thus comprise the first DNA template, the second oligonucleotide, a DNA polymerase, deoxyribonucleoside triphosphates and the appropriate reaction buffer as described herein.
- the reaction may be allowed to proceed at selected temperatures and for sufficient time to enable the first template (DNA template or RNA template) and the second oligonucleotide (though the hybridizing portion) to anneal and the 3'-end of the first template.
- the first template may be extended with the second oligonucleotide serving as the template and the 3'-end of the first template as primer extension. Extension of the second oligonucleotide may also proceed concomitantly.
- the present invention more particularly relates to a method for adding a terminal sequence tag to a target nucleic acid molecule, the method may comprise the step of; a. contacting the target nucleic acid molecule with a first oligonucleotide which may comprise; a) a first 5'-overhanging portion which comprise a first sequence tag; b) a first hybridizing portion which may be able to hybridize to the target nucleic acid molecule, and; c) a blocked 3'-end, under conditions which may allow hybridization of the first hybridizing portion with the target nucleic acid molecule, b. extending the target nucleic acid molecule to generate a first template which may comprise a complementary first sequence tag, c.
- the present invention also more particularly relates to a method for adding a terminal sequence tag to a plurality of target nucleic acid molecules, the method may comprise, for example; a.
- contacting the plurality of target nucleic acid molecules with a plurality of first oligonucleotides each may comprise; a) a first 5'-overhanging portion which may comprise a first sequence tag; b) a first hybridizing portion which may be able to hybridize to the plurality of target nucleic acid molecules, and; c) a blocked 3'-end, under conditions which may allow hybridization of the first hybridizing portion with the plurality of target nucleic acid molecules, b. extending the plurality of target nucleic acid molecules to generate a plurality of first templates which may comprise a complementary first sequence tag, c.
- a (a mixture of) second oligonucleotides which may comprise a) a second 5'-overhanging portion which may comprise a second sequence tag; and b) a second hybridizing portion which may comprise a first sequence tag, under conditions which may allow hybridization of the second hybridizing portion with the plurality of first templates and; d. extending wholly or partially the plurality of first templates and second oligonucleotides to generate a plurality of second templates.
- the target nucleic acid molecule is a sense RNA (e.g., mRNA)
- the RNA may be converted into a cDNA prior to performing methods of the present invention.
- the methods of the present invention further comprise a step of converting a mRNA into a (complete or partial) cDNA prior to adding a (terminal) sequence tag.
- a step of removing or inactivating the first oligonucleotide (or plurality of first oligonucleotides) before contacting the first template with a second oligonucleotide may also be performed.
- the first and second 5'-overhanging portion may be identical or different. It is to be understood that the method of the present invention may "comprise” the indicated steps or may “consist essentially” of the indicated steps or even may “consist” of the indicated steps.
- the term "consisting essentially of means that the method consists of the indicated steps and comprises other steps which does not affect in a significant manner, the working of the methods.
- Methods for adding first, second or first and second sequence tag to a target nucleic acid molecule are thus encompassed by the present invention.
- One advantage of the methods of the present invention is that it may allow, for example, terminal tagging of nucleic acids molecules such as a full length cDNA.
- the sequence tag which is introduced may be used as a primer binding site for subsequent amplification of the DNA molecule and/or sequencing of the DNA molecule and therefore provides means for identification and cloning of the 5'-end or the complete sequence of previously unknown mRNAs sequence.
- sequence tag introduced by the method of the present invention is a RNA polymerase promoter or comprise a RNA polymerase promoter
- linear amplification of RNA from tagged cDNA may therefore occur and permits quantification of the relative abundance of the initial target (e.g., mRNA) in a sample.
- a tag at the 5'-end of a nucleic acid may also be advantageous in the generation of full length or partial cDNA libraries and therefore allows identification of the complete or partial sequence of RNA species or differentially expressed RNA species.
- the present invention additionally provides methods for generating RNA from the template described herein.
- the method may comprise, for example, providing the first template to which an oligonucleotide comprising a RNA polymerase promoter is annealed, with a RNA polymerase enzyme and RNA polymerase reagents under condition suitable for RNA polymerization.
- a first template may be suitable for RNA transcription if an oligonucleotide comprising a RNA polymerase promoter has been annealed to it, thus making a double-stranded RNA polymerase promoter.
- the method may also comprise, for example, providing the second template comprising a double-stranded RNA polymerase promoter (and other regulatory regions) with a RNA polymerase enzyme and RNA polymerase reagents under condition suitable for RNA polymerization.
- the method described herein may generate a first DNA template and a second DNA template which may have, for example, a double-stranded RNA polymerase promoter located at a 3'-end of the double-stranded second DNA template (which is at least partially or totally double-stranded, or at least double-stranded in the promoter region), thereby producing an antisense RNA using the method of generating RNA described herein.
- a double-stranded RNA polymerase promoter located at a 3'-end of the double-stranded second DNA template (which is at least partially or totally double-stranded, or at least double-stranded in the promoter region), thereby producing an antisense RNA using the method of generating RNA described herein.
- the method described herein may generate a first DNA template and a second DNA template which may have, for example, a double-stranded RNA polymerase promoter located at a 5'-end of the double-stranded second DNA template (which is at least partially or totally double-stranded, or at least double-stranded in the promoter region), thereby producing a sense RNA using the method of generating RNA described herein.
- a double-stranded RNA polymerase promoter located at a 5'-end of the double-stranded second DNA template (which is at least partially or totally double-stranded, or at least double-stranded in the promoter region), thereby producing a sense RNA using the method of generating RNA described herein.
- the present invention relates in an additional aspect thereof, to a method for generating RNA from a nucleic acid comprising a suitable second template (i.e., comprising a RNA polymerase promoter) as described herein, the method may comprise providing the nucleic acid with a RNA polymerase enzyme and RNA polymerase reagents under condition suitable for RNA polymerization.
- a suitable second template i.e., comprising a RNA polymerase promoter
- the present invention also relates to a method for generating RNA from a vector described herein, the method may comprise providing the vector with a RNA polymerase enzyme and RNA polymerase reagents under condition suitable for RNA polymerization. Additionally, the present invention relates to a method for generating RNA from a cell as described herein, the method may comprise providing the cell with a RNA polymerase enzyme and RNA polymerase reagents under condition suitable for RNA polymerization.
- the nucleic acid target used in the method of the present invention may be a single-stranded positive DNA molecule thereby introducing a double-stranded RNA polymerase promoter at a region located at a 3'- end of the second template.
- the nucleic acid target used in the method of the present invention may be a single-stranded negative DNA or a cDNA molecule thereby introducing a double-stranded RNA polymerase promoter at a region located at a 5'-end of the second template.
- the nucleic acid target used in the method of the present invention may be a sense RNA molecule thereby introducing a double-stranded RNA polymerase promoter at a region located at a 3'- end of the second template.
- the nucleic acid target used in the method of the present invention may be an anti-sense RNA molecule thereby introducing a double-stranded RNA polymerase promoter at a region located at a 5'-end of the second template.
- the RNA polymerase suitable for the methods of the present invention may be any enzyme capable of recognizing the double-stranded promoter and specifically initiating RNA synthesis at the defined initiation site within close proximity to the promoter. Preparations comprising the RNA polymerase may be relatively free of contaminating agents with DNase or RNase activities. In addition the RNA polymerase may be capable of synthesizing several copies of RNA per functional copy of DNA template in a desirable period of time. In accordance with the present invention, the RNA polymerase may be selected from the group consisting of, and without limitation, the bacteriophage 11 RNA polymerase, the phage T3 RNA polymerase, the Salmonella phage sp6 RNA polymerase etc. It is understood by those skilled in the art that the use of alternative RNA polymerases will involve changes to the sequence of the promoter template according to the specificity of the particular RNA polymerase.
- the transcription reaction may comprise the desirable concentrations of cofactors and nucleoside triphosphates for RNA synthesis using the particular RNA polymerase.
- the transcription reaction may be performed under the conditions of pH, ionic strength and temperature that are desirable for the enzyme which is used. Such reaction conditions are known to those skilled in the art and are usually provided by the manufacturer.
- RNA may thus be synthesized from the second DNA template comprising a double- stranded RNA polymerase promoter sequence generated by the method of the present invention. Therefore, RNA may be synthesized from the second DNA template having a double-stranded promoter sequence.
- RNA synthesis may occur to the extent that at least the RNA polymerase region is double-stranded. It is therefore understood herein that RNA may be synthesized from a template having a double stranded promoter sequence and a single- or double-stranded remaining sequence.
- RNA may be synthesized from the first or second DNA template having a double-stranded promoter sequence by using an RNA polymerase that is specific to the particular promoter sequence.
- the reaction may comprise, for example, the DNA templates (e.g., first DNA template having a double- stranded promoter sequence or second DNA template having a double-stranded promoter), a RNA polymerase buffer [40 mM Tris-HCI (pH 7.9), 6 mM MgCI 2 , 2 mM spermidine, 10 mM DTT] supplemented with an equimolar mixture of ATP, UTP, GTP and CTP incubated at about 37 0 C for a specified period.
- the DNA templates e.g., first DNA template having a double- stranded promoter sequence or second DNA template having a double-stranded promoter
- a RNA polymerase buffer [40 mM Tris-HCI (pH 7.9), 6 mM MgCI 2
- RNA produced by the method of the present invention may further be reverse transcribed and/or amplified to generate, for example, cDNA libraries.
- Another aspect of the invention provides a method for synthesizing RNA from DNA molecules.
- This method comprises forming first DNA templates by adding a terminal sequence tag to the DNA molecules; forming first DNA templates having a double- stranded promoter sequence and/or forming second DNA templates which may be at least double-stranded in the promoter region and synthesizing RNA from the first or second DNA templates having a double-stranded promoter sequence.
- the first DNA templates having a double-stranded promoter sequence may be formed by contacting the first DNA templates with oligonucleotides containing the sequence tag, a promoter template, a random sequence and a blocked 3' terminus, under conditions such that, the random sequence anneals with the DNA molecules and the DNA molecules are extended using the sequence tag and promoter as template.
- the second DNA templates having a double-stranded promoter sequence may be formed by contacting the first DNA templates without a promoter with a second oligonucleotide containing the sequence tag complement to the tag sequence contained in the first DNA templates and a promoter sequence template, under conditions such that, the first DNA templates anneal with the sequence tag complement of the second oligonucleotide and are extended using the promoter sequence as template.
- the second oligonucleotide may contain a blocked 3' terminus.
- a terminal sequence tag may be added to DNA molecules by contacting with a mixture of oligonucleotides, each having a sequence tag, a random sequence and a blocked 3' terminus, under conditions such that, the random sequence anneals with the DNA molecules and the DNA molecules are extended using the sequence tag as template.
- DNA molecules may be formed by contacting a mixture containing mRNA with a primer having a terminal sequence complementary to the mRNA, under conditions such that, the terminal sequence of the primer anneals with the mRNA and is extended using the mRNA as template.
- Another aspect of the invention provides a method for synthesizing first RNA templates having a double-stranded promoter sequence comprising contacting the RNA molecules with oligonucleotides containing the sequence tag, a promoter template, a random sequence and a blocked 3' terminus, under conditions such that, the random sequence anneals with the RNA molecules and the RNA molecules are extended using the sequence tag and promoter templates as template.
- the second RNA templates having a double-stranded promoter sequence may be formed by contacting the first RNA templates without a promoter with a second oligonucleotide containing the sequence tag complement to the tag sequence contained in the first RNA templates and a promoter sequence template, under conditions such that, the first RNA templates anneal with the sequence tag complement of the second oligonucleotide and are extended using the promoter sequence as template.
- the second oligonucleotide may contain a blocked 3' terminus.
- a terminal sequence tag may be added to DNA molecules by contacting with a mixture of oligonucleotides, each having a sequence tag, a random sequence and a blocked 3' terminus, under conditions such that, the random sequence anneals with the DNA molecules and the DNA molecules are extended using the sequence tag as template.
- DNA molecules may be formed by contacting a mixture containing mRNA with a primer having a terminal sequence complementary to the mRNA, under conditions such that, the terminal sequence of the primer anneals with the mRNA and is extended using the mRNA as template.
- DNA sequences may be amplified using standard techniques which are known by those of skill in the art.
- the first or second template (partially or wholly double-stranded) generated by the methods of the present invention may thus be amplified by using at least one primer which is complementary to a sequence contained in the template.
- One of the sequence which may serve as a primer binding site is, for example, a sequence tag introduced by the method of the present invention.
- DNA templates may be amplified in vitro using, for example, technologies such as PCR 1 NASBA and SDA.
- the primers may contain, for example, a restriction endonuclease site in order to aid in cloning of the amplified DNA templates.
- this tag when the sequence tag is introduced in a full length cDNA generated by reverse transcription of an unknown mRNA, this tag (tag site) may subsequently be used for amplification, sequencing, transcription (transcription -coupled translation) and therefore identification of the complete sequence of the unknown target nucleic acid as well as its amino acid sequence.
- this tag when a sequence tag is introduced in a full length cDNA generated by reverse transcription of a partially known mRNA, this tag (tag site) may subsequently be used to isolate and identify the complete sequence of the target nucleic acid molecule.
- another aspect of the invention provides a method for amplifying terminal sequences of DNA molecules comprising: forming first DNA templates by adding a terminal sequence tag to the DNA molecules; forming double-stranded DNA templates by extending a first primer; and amplifying the DNA templates by extending the first primer and a second primer.
- the double-stranded DNA templates may be formed by contacting the first DNA templates with a first primer having a sequence complementary to the sequence tag, under conditions such that, the sequence of the primer anneals with the sequence tag of the first DNA templates and is extended.
- the DNA templates may be amplified by contacting with the first primer and a second primer containing a sequence complementary to a sequence from the complementary DNA strand to the first DNA templates, under conditions such that the primers anneal to complementary templates and are extended.
- a terminal sequence tag may be added to DNA molecules by contacting with a mixture of oligonucleotides, each having a sequence tag, a random sequence and a blocked 3' terminus, under conditions such that, the random sequence anneals with the DNA molecules and the DNA molecules are extended using the sequence tag as template.
- DNA molecules may be formed by contacting a mixture containing mRNA with a primer having a terminal sequence complementary to the mRNA, under conditions such that, the terminal sequence of the primer anneals with the mRNA and is extended using the mRNA as template.
- the present invention also relates to kits and reagents for carrying out the present invention.
- the present invention therefore relates in one aspect thereof to a first template and second template generated by the methods described herein as well as the RNA generated by their transcription.
- the second template may be at least partially double-stranded or totally double-stranded.
- the second template may comprise a double-stranded second sequence tag region.
- the double-stranded second sequence tag region may be, for example, a double-stranded promoter region, a double- stranded restriction endonuclease region, etc.
- the double-stranded promoter region may comprise, for example, a double-stranded RNA polymerase promoter.
- the present invention relates to a nucleic acid molecule comprising a first template or a second template generated by the methods described herein.
- the present invention relates to a vector comprising a first template or a second template generated by the methods described herein.
- the present invention relates to a cell (e.g., an isolated cells, a cell line, such as, for example, a mammalian cell, an insect cell, an animal cell, etc.) which may comprise the first template, second template, nucleic acid or vector described herein.
- a cell e.g., an isolated cells, a cell line, such as, for example, a mammalian cell, an insect cell, an animal cell, etc.
- kits include one or more reagents used in the methods described herein.
- Exemplary embodiments of kits are those which may include, for example, a container comprising at least one oligonucleotide described herein.
- a kit may include, for example, an oligonucleotide having a promoter sequence.
- a kit may also include one or more enzymes for polymerizing ribonucleotides and/or deoxyribonucleotides, such as a DNA polymerase, RNA polymerase, a reverse transcriptase.
- a kit may also comprise an oligonucleotide complementary to mRNA molecules (e.g., oligo(dT), or containing a specific complementary sequence).
- the present invention therefore relates in one aspect thereof to a kit which may comprise a first oligonucleotide or the plurality of first oligonucleotides described herein.
- the kit may also further comprise a second oligonucleotide as described herein.
- the present invention also relates to a kit which may comprise a second oligonucleotide as described herein.
- the present invention relates more particularly to a kit which may comprise: (a) a first oligonucleotide as described herein (b) a DNA polymerase and reagents for extending the 3'- ends of the nucleic acid molecules; (c) size selection columns and buffers for removal of unused first oligonucleotide; and (d) instructions for hybridizing the first oligonucleotide to the target nucleic molecule, extending the target nucleic acid molecule with the DNA polymerase using the first oligonucleotide as template, and creating first DNA or RNA templates with, for example, double-stranded regions.
- the kit may further comprise a second oligonucleotide as described herein and instructions to use the second oligonucleotide as template to form second DNA templates or second RNA templates, containing a sequence of interest such as, for example, a promoter.
- the kit may further comprise a reverse transcriptase, at least one enzyme for RNA hydrolysis and an oligonucleotide complementary to mRNA molecules (e.g., oligo(dT)). Further in accordance with the present invention, the kit may further comprise a RNA polymerase matching a functional promoter and reagents, and cofactors for in vitro RNA synthesis from the promoter.
- the kit may further comprise a first primer which may correspond to the sequence tag and at least one second primer for exponential amplification of double stranded second DNA template, wherein the primers anneal to the a strand of the template which complementary and are extended repeatedly.
- the present invention relates to a composition which may comprise a target nucleic acid molecule having annealed at its 3'-end thereof a first oligonucleotide which may comprise a) a 5'-overhanging portion substantially non- hybridized to the target nucleic acid molecule, the 5'-overhanging portion may comprise a sequence tag; b) an hybridizing portion hybridized to the target nucleic acid molecule, and; c) a blocked 3'-end or an unblocked 3'-end.
- the nucleic acid molecule may be DNA or a RNA or a DNA/RNA hybrid, etc.
- the sequence tag may be a promoter sequence.
- the present invention relates in one aspect thereof, to methods kits and reagents as described in U.S. patent application No. 11/000,958 published on July 14, 2005 under No. US 2003/0153333 A1.
- the present invention also relates to improvements to the methods, kits and reagents described above.
- the present invention relates in an aspect therefore to an oligonucleotide which may comprises, for example, a) a 5'-overhanging portion which may comprise a first sequence tag; b) an hybridizing portion which may be able to hybridize to the target nucleic acid molecule, the hybridizing portion may comprise a ribonucleic acid section and; c) a blocked 3'-end.
- the present invention also relates to a kit comprising an oligonucleotide as described herein.
- the kit may further comprise an enzyme and/or one or more other reagents or instructions useful to carry out the method of the present invention.
- the present invention relates in an additional aspect thereof, to a method for adding a terminal sequence tag to a target nucleic acid molecule
- the method may comprise, for example, i. contacting the target nucleic acid molecule with an oligonucleotide which may comprise: a) a 5'-overhanging portion which include a first sequence tag; b) an hybridizing portion which may be able to hybridize to the target nucleic acid molecule, the hybridizing portion may comprise a ribonucleic acid section and; c) a blocked 3'-end, under conditions which may allow hybridization of the hybridizing portion with the target nucleic acid molecule, ii.
- extending the target nucleic acid molecule to generate a first template which may comprise a complementary first sequence tag, iii. removing (e.g., enzymatically removing) the ribonucleic acid section of the oligonucleotide to generate a primer extension site and; iv. extending the oligonucleotide to generate a second template.
- the present invention also relates in a further aspect to a method for adding a terminal sequence tag to a plurality of target nucleic acid molecules, the method may comprise, for example, i. contacting the plurality of nucleic acid molecules with a plurality of oligonucleotides each of which may comprise: a) a 5'-overhanging portion which may comprise a first sequence tag; b) an hybridizing portion which may be able to hybridize to a target nucleic acid molecule, the hybridizing portion may comprise a ribonucleic acid section and; c) a blocked 3'-end, under conditions which may allow hybridization of the hybridizing portion with the target nucleic acid molecule, ii.
- extending the plurality of target nucleic acid molecules to generate a plurality of first templates which may comprise a complementary first sequence tag, iii. removing (e.g., enzymatically removing) the ribonucleic acid section of the oligonucleotide to generate a primer extension site and; iv. extending the oligonucleotide to generate a plurality of second templates.
- the first sequence tag may be selected, for example, from the group consisting of a promoter sequence, an endonuclease restriction site, an hybridization site and any combination thereof.
- the removal step may be performed with the help of an enzyme such as, for example, a RNase.
- an enzyme such as, for example, a RNase.
- the hybridizing portion may comprise a) a random sequence or b) a nucleic acid sequence substantially complementary (e.g., 80 to 100% complementarity over the entire sequence or portion of sequences) to a portion located at a 3'-end of a target nucleic acid molecule (with respect to the 5'- > 3' direction).
- the hybridizing sequence of the improved oligonucleotide may thus comprise in part or as a whole, an equal representation of ribonucleotides G, A, U and C to form
- Wobble bases such as inosine (I) may also be used instead of standard bases at any of the positions.
- one or more of the ribonucleotides may be chemically modified while still maintaining a functional activity as a substrate for ribonuclease.
- the replacement of part deoxyribonucleotides with ribonucleotides in the oligonucleotide is not limited to only the random sequence or 3'-complementary sequence and may include any number of nucleotides 5' and 3' of these sequences.
- This modification of the oligonucleotides sequence tag may facilitate the synthesis of fully double-stranded "second DNA templates" without the need for use of a second oligonucleotide containing a promoter as described above.
- this improvement provides for a more homogenous terminal tagging process with the elimination of a nucleic acids clean-up step.
- the 3'-proximal random sequence may be any number of nucleotides in length but preferably between 4 and 9 (or 4 to 15). Also, the 3' terminus of the modified oligonucleotides may be chemically blocked as described in the EXAMPLE section.
- reaction conditions may be applied such that, the random sequences of the oligonucleotides may anneal with the nucleic acid molecules and the nucleic acid molecules may be extended using as template the promoter containing sequence tag of the oligonucleotides.
- the oligonucleotides and nucleic acid molecules may be allowed to anneal by heating a mixture of these two components at an elevated temperature (> 37°C) for a period of time and then incubating at a temperature suitable for enzymatic extension of the nucleic acid molecules, depending on the nature of the enzyme used.
- the nucleic acid molecules may be extended as described above by using a DNA polymerase, which may be any enzyme capable of synthesizing DNA by extending a DNA or RNA primer using either a RNA or DNA template.
- the DNA polymerase may be substantially free of exonuclease activities, either 3' to 5' or 5' to 3', and preparations containing the DNA polymerase may be relatively free of agents capable of nucleic acid hydrolysis. Examples of DNA polymerase that may be AMV and M-MLV reverse transcriptases, and Tth DNA polymerase.
- the nucleic acid molecules may be composed of DNA.
- the resulting "first DNA templates" may have a 3' promoter sequence and sequence tag (plus specific sequence tag) that is complementary (at least partially complementary) to the sequence tag contained in the oligonucleotide mixture (see schematic of Fig. 9a for illustration).
- the resulting "first DNA templates” may comprise a 3' promoter sequence which may also serve as the sequence tag (minus specific sequence tag) that is complementary to the sequence tag contained in the oligonucleotide mixture (see schematic of Fig. 9b for illustration).
- the resulting "first DNA templates” are now partially double-stranded comprising a double-stranded promoter sequence with the oligonucleotide template strands still blocked at the 3' terminus (see schematic of Fig. 9a and 9b for illustration).
- RNA may be synthesized from DNA molecules by forming first DNA templates having a double- stranded promoter sequence, forming second DNA templates having a double- stranded promoter sequence and synthesizing RNA from the first or second DNA templates having a double-stranded promoter sequence.
- the nucleic acid molecules may be composed of RNA, wherein the resulting "first RNA templates" have a 3' promoter sequence and sequence tag (plus specific sequence tag) that is complementary to the sequence tag contained in the oligonucleotide mixture (see schematic of Fig. 10a for illustration).
- the resulting "first RNA templates” may have a 3 1 promoter sequence which may also serve as the sequence tag (minus specific sequence tag) that is complementary to the sequence tag contained in the oligonucleotide mixture (see schematic of Fig. 10b for illustration).
- the resulting "first RNA templates” are now partially double-stranded comprising a double-stranded promoter sequence with the oligonucleotides template strands still blocked at the 3' terminus.
- completely double-stranded "second DNA templates" having a double-stranded promoter sequence may be formed by applying reaction conditions such that, the ribonucleotide random sequence of the oligonucleotides sequence tag now part of an RNA:DNA hybrid may become hydrolyzed by a specific ribonuclease such as, ribonuclease H, thereby releasing the 3' terminus blocking group.
- the resulting oligonucleotides sequence tag of the double- stranded promoter of the "first DNA templates” may now contain an unblocked 3" terminus with a 3' OH group capable of extension by DNA polymerization.
- An RNA- or DNA-directed DNA polymerase present in the reaction under the appropriate conditions may thus extend the unblocked oligonucleotides sequence tag to synthesize the completely double-stranded "second DNA templates".
- RNA may be synthesized from a DNA template having a double-stranded promoter sequence by using an RNA polymerase that is specific to the particular promoter sequence (see schematic of Fig. 11 for illustration).
- the reaction comprises the DNA template, a RNA polymerase buffer [40 mM Tris-HCI (pH 7.9), 6 mM MgCI 2 , 2 mM spermidine, 10 mM DTT] supplemented with an equimolar mixture of ATP, UTP, GTP and CTP incubated at 37 0 C for a specified period.
- RNA synthesis from a completely double-stranded "second DNA templates" having a double-stranded promoter sequence may proceed more efficiently than double- stranded first DNA templates containing only a double-stranded promoter sequence or partially double-stranded second DNA template.
- the described improvement of the selective terminal tagging method involves the use of a single oligonucleotides sequence tag instead of two separate oligonucleotides for the addition of firstly, the sequence tag and secondly, the promoter sequence in order to synthesize fully double-stranded "second DNA templates".
- modified oligonucleotides sequence tag may hybridize at internal sites along the nucleic acids molecules and become extended following RNase H digestion and DNA polymerization, the resulting molecules will not contain a double-stranded promoter sequence required for efficient RNA synthesis and thus no RNA from these species may be generated.
- a second round of tagging may be performed on RNA (or cDNA when reverse transcription occurs) produced by the methods of the present invention to further amplify target nucleic acid molecules.
- the RNA synthesized from the "first or second DNA templates" may contain the specific sequence tag sequence at its 5' terminus.
- the modified oligonucleotides comprise only a promoter sequence (and no other tag), a random sequence and a blocked 3'-terminus are used for the methods of the present invention (see schematic of Fig. 9b for illustration)
- the RNA synthesized from the "first or second DNA templates” will not contain a specific sequence tag sequence at the 5' terminus. Therefore, the promoter sequence of the "first or second DNA templates" may itself serve as a specific sequence tag for primer- directed DNA synthesis.
- the present invention also relates to templates, RNA, composition of template annealed with oligonucleotide, etc. generated using the methods of the present invention. Definitions
- sequence tag is a general term used to refer to either a "complementary” sequence tag or a “sense” sequence tag or to both.
- sequence tag is not intended to be restricted to a specific sense and therefore refers to either a "complementary” sequence tag or a “sense” sequence tag or to both.
- the term "at least one oligonucleotide” may refer to either a numerical value or to an oligonucleotide species or sequence such as in the expression "at least one oligonucleotide species”.
- substantially non-hybridizable means that a sequence does not hybridize with another one in a significant manner or in a manner affecting the way the invention is carried out.
- a first DNA template comprising a double-stranded region able to be recognized by restriction endonuclease is referred herein as being "activated".
- sequence identity relates to (consecutive) nucleotides of a nucleotide sequence which with reference to an original nucleotide sequence. The identity may be compared over a region or over the total sequence of a nucleic acid sequence.
- identity may be compared, for example, over a region of 3, 4, 5, 10, 19, 20 nucleotides or more (and any number there between). It is to be understood herein that gaps of non-identical nucleotides may be found between identical nucleic acids.
- an oligonucleotide may have 100% identity with another oligonucleotide over a portion thereof. However, when the entire sequence of both oligonucleotides is compared, the two oligonucleotides may have 50% of their overall (total) sequence identical to one another.
- Oligonucleotides of the present invention or portion thereof having from about 80 to 100% sequence identity or from about 90 to 100% sequence identity or from about 95 to 100% sequence identity with an original oligonucleotide are encompassed herewith. It is known by those of skill in the art, that an oligonucleotide having from about 80% to 100% identity may function (e.g., anneal to a substantially complementary sequence) in a manner similar to an original oligonucleotide and therefore may be used in replacement of an original oligonucleotide.
- an oligonucleotide (a nucleic acid sequence) may comprise or have from about 80% to 100% identity with an original oligonucleotide over a defined region and may still work as efficiently or sufficiently to achieve tagging of a target nucleic acid molecule.
- Percent identity may be determined, for example, with n algorithm GAP, BESTFIT, or FASTA in the Wisconsin Genetics Software Package Release 7.0, using default gap weights.
- sequence complementarity refers to (consecutive) nucleotides of a nucleotide sequence which are complementary to a reference (original) nucleotide sequence. The complementarity may be compared over a region or over the total sequence of a nucleic acid sequence.
- Oligonucleotides of the present invention or portion thereof having from about 80 to 100% sequence complementarity or from about 90 to 100% sequence complementarity or from about 95 to 100% sequence complementarity with an original oligonucleotide are encompassed herewith. It is known by those of skill in the art, that an oligonucleotide having from about 80% to 100% complementarity with an original sequence may anneal to that sequence in a manner sufficient to carry out the methods of the present invention.
- an oligonucleotide (a nucleic acid sequence) may comprise or have from about 80% to 100% complementarity with an original oligonucleotide and may still anneal with the original sequence in a manner sufficient to achieve tagging of a target nucleic acid molecule.
- first template includes a “first DNA template” and a “first RNA template”.
- first DNA template means a DNA molecule, such as, for example a negative (uncoding) strand (3'-> 5') or a positive (coding) strand (5'-> 3 1 ) and which comprises at its 3'-end, a sequence tag such as a complementary first sequence tag.
- the complementary first sequence tag contained within the "first DNA template” may be substantially complementary to the first sequence tag contained in the first oligonucleotide used in an exemplary method of the present invention (see schematic of Figure 2 for illustration of tagging of a cDNA molecule).
- first RNA template means a RNA molecule, such as, for example, a sense or anti-sense RNA and which may comprise at its 3'-end, a sequence tag.
- sequence tag contained within the "first RNA template” may be a complementary first sequence tag which may be (substantially) complementary to the first sequence tag contained in the first oligonucleotide used in an exemplary method of the present invention.
- the first RNA templates formed by the present methods may thus comprise a composite of deoxy- and ribonucleotides (see schematic of Figure 3 for illustration of terminal tagging of a sense RNA), which may be effected, for example, by RNA-directed DNA polymerase such as but not limited to AMV reverse transcriptase may be used.
- second template includes a “second DNA template” and a “second RNA template”.
- second DNA template means an at least partially double- stranded DNA molecule comprising at an end thereof, a sequence tag (sequence of interest) such as a second (and in some circumstances, a first) sequence tag (see schematic of Figure 4 for illustration of a second DNA template) .
- the "second DNA template may comprise at its 5'-end (with respect to the coding sequence) and from a 5'-> 3' direction 1 ) a second sequence tag (sequence of interest) and; 2) a first sequence tag. It is also to be understood herein that when the "first DNA template” is a single-stranded coding strand of a DNA molecule, the "second DNA template may comprise at its 3'-end (with respect to the coding sequence) and from a 5'-> 3' direction 1) a first sequence tag and 2) second sequence tag (a sequence of interest).
- second RNA template means a RNA molecule, such as, for example, a sense or anti-sense RNA which comprises at its 3'-end and from a 5'-> 3' direction 1) a first sequence tag and 2) second sequence tag (a sequence of interest) or a complement of a sequence of interest.
- a first primer When a linear amplification of a nucleic acid is required, it is desirable to have a first primer with a blocked 3'-end. However, in some circumstances, such as for cloning purposes, a blocked 3'-end is not always necessary.
- any specified range or group is to be understood as a shorthand way of referring to each and every member of a range or group individually as well as each and every possible sub-ranges or sub-groups encompassed therein; and similarly with respect to any sub-ranges or sub-groups therein.
- any specified range or group is to be understood as a shorthand way of referring to each and every member of a range or group individually as well as each and every possible sub-ranges or sub-groups encompassed therein; and similarly with respect to any sub-ranges or sub-groups therein.
- the methods of the present invention each include each and every individual steps described thereby as well as those defined as positively including particular steps or excluding particular steps or a combination thereof; for example an exclusionary definition for a method of the present invention, may read as follows: "provided that when the nucleic acid target is a mRNA, the hybridizing portion is preferably not a oligo(dT) sequence or if an oligo(dT) is used, the 3'-end is blocked, etc.
- FIG. 1 shows a schematic illustration of the synthesis of cDNA molecules from mRNA molecules
- FIG. 2 shows a schematic illustration of the synthesis of first DNA templates comprising a sequence tag from cDNA molecules
- FIG. 3 shows a schematic illustration of the synthesis of first RNA templates comprising a sequence tag from RNA molecules
- FIG. 4 shows a schematic illustration of the synthesis of second DNA templates containing a promoter sequence and transcription of RNA from second DNA template
- FIG. 5 shows agarose gel electrophoretic analysis of the products of transcription reactions from second DNA template with or without a terminal sequence tag, as detected by ethidium bromide staining (A) or by blot hybridization with 32 P labeled cDNA probes to GAPDH (B) and ⁇ -actin (C);
- FIG. 6 shows agarose gel electrophoretic analysis of products from PCR amplification of tagged (or untagged) second DNA template prepared with or without a terminal sequence tag and a common forward primer (first primer) in combination with gene specific reverse primers (second primers) for GAPDH and actin, as detected by ethidium bromide staining (A) or by blot hybridization with 32 P labeled cDNA probes to GAPDH (B) and ⁇ -actin (C);
- FIG. 7 shows agarose gel electrophoretic analysis of the products of transcription reactions from cDNA prepared with a terminal sequence tag, as detected by ethidium bromide staining (A) or by blot hybridization with 32 P labeled cDNA probes to Cathepsin K (B);
- FIG. 8 shows a plot of the hybridization signal of the probe to Cathepsin K (vs. Precursor/Osteoclast RNA ratios), quantified by scintillation counting of bands excised from the hybridized blot shown in FIG. 7B, versus the fraction of osteoclast RNA in the RNA mixture;
- FIG. 9a is a schematic illustrating the terminal tagging of a cDNA molecule using an improved method according to an embodiment of the present invention.
- FIG 9b is a schematic illustrating the terminal tagging of a cDNA molecule using an improved method according to an further embodiment of the present invention
- FIG 10a is a schematic illustrating the terminal tagging of a RNA molecule using an improved method according to an additional embodiment of the present invention
- FIG 10b is a schematic illustrating the terminal tagging of a RNA molecule using an improved method according to a further embodiment of the present invention.
- FIG 11 is a schematic illustrating linear RNA amplification from the second DNA template according to yet an additional embodiment of the invention, and;
- FIG 12 panel A is a photograph of an agarose gel showing the electrophoretic profile of transcription reactions products from tagged target generated by standard and improved methods described herein
- Panel B is a Northern blot of the same product, visualized with a radioactive probe.
- the products obtained by the methods of the present invention may have a variety of utilities including, without limitation, cloning of known or unknown target nucleic acid molecule, the generation of hybridization probes, the construction of cDNA libraries, and the analysis and identification of the terminal sequence or complete nucleic acid sequences (and amino acid sequence) of the target nucleic acid molecules. More particularly, methods of the present invention allow linear amplification of a target nucleic acid for determination of the relative abundance of the target amongst other nucleic acid molecules.
- RNA from mouse brain was repurified using the RNeasy procedure (Qiagen).
- the mRNA population contained in 4 ⁇ g of total RNA was used for making first-strand cDNA in a standard cDNA synthesis reaction containing 7.5 ⁇ M oligo dT primer (Seq. ID. No.
- the purified first-strand cDNA molecules were then divided into 2 equal aliquots and dried.
- 1.5 nmol (7.5 ⁇ L) of the oligonucleotide sequence tag (Seq. ID. No. 2; GACGAAGACAGTAGACAN X (N(2'-O-Methyl))(3'-C3 propyl spacer) note that x is 6) was added and to the second aliquot, 7.5 ⁇ L water. Both aliquots were incubated at 65 0 C for 5 min and then at 37°C for 10 min.
- each aliquot was adjusted to 20 ⁇ L by adding components of a DNA synthesis reaction, at final concentrations of 1 mM Tris-HCI (pH 7.5), 0.5 mM MgCI 2 , 0.75 mM DTT, 33 ⁇ M dATP, 33 ⁇ M dGTP, 33 ⁇ M dCTP, 33 ⁇ M TTP and 0.5 units/ ⁇ L Klenow fragment (3' to 5' exo ' ) (New England Biolabs). The reactions were incubated for an additional 60 minutes at 37°C and then terminated with the addition of phenol.
- the first DNA templates formed in reaction 1 was then purified from any excess sequence tag oligonucleotide by size selection (Amersham) in a final volume of approximately 40 uL.
- the first-strand cDNA molecule from reaction 2 was similarly purified although it did not contain any oligonucleotide sequence tag.
- the random sequence at each nucleotide position was synthesized from an equal mixture of the four phosphoramidites by Trunk Biotechnologies (San Diego, CA) and the oligonucleotide was PAGE purified. The length of the random portion in each example is seven.
- the DNA templates from each of the 2 reactions in Example 1 were used for priming DNA synthesis using a second oligonucleotide template containing a 5' T7 promoter sequence (italicized) and a 3' sequence tag (Seq. ID. No. 3; AATTCTAATACGACTCACTATAGGGAGACGAAGACAGTAGACA) similar to the sequence tag contained in the first oligonucleotide to form second DNA templates containing a T7 promoter sequence.
- the DNA synthesis reactions (50 uL) contained the respective DNA templates, 5 pmoles second oligonucleotide template (Seq. ID. No.
- DNA synthesis could as well be primed from the second oligonucleotide template using the first DNA templates as template in the same reaction (see schematic of Figure 4 for illustration) to form completely double-stranded second DNA templates.
- the resulting DNA templates from both reactions were purified by size selection (Amersham) and transcribed in vitro.
- Each in vitro transcription reaction (40 ⁇ L) comprised the respective DNA templates, 40 mM Tris-HCI (pH 7.9), 6 mM MgCI 2 , 2 mM spermidine, 10 mM DTT, 0.5 mM ATP, 0.5 mM GTP, 0.5 mM CTP, 0.5 mM UTP and 4 ⁇ L T7 RNA polymerase (Ambion).
- the reactions were incubated at 37°C for at least 2 hours, digested with DNase I at 37°C for 30 minutes, phenol extracted and purified.
- Lane 1 contains 200 ng of neat total RNA from mouse brain
- Lane 2 contains a 4- ⁇ L aliquot of the transcription reaction from the second DNA templates containing the T7 promoter sequence
- Lane 3 contains a 4- ⁇ L aliquot of the transcription reaction from DNA templates prepared without the addition of the oligonucleotide sequence tag (cDNA molecules) (Seq. ID. No. 2).
- RNA of various sizes (a RNA smear ranging from -300 bp to -1650 bp based on the 1 Kb Plus DNA ladder (InVitrogen)), as expected from a library of cDNA molecules, were synthesized from the second DNA templates whereas, in Lane 3, no such RNA was observed.
- FIG. 5 contains the following:
- RNA In vitro transcribed RNA (5 ⁇ g) generated in Example 2 containing the oligonucleotide sequence tag at its 5' proximal end was reverse transcribed in a standard cDNA synthesis reaction (In Vitrogen) and the resulting first-strand cDNA was purified and reconstituted in 20 ⁇ L H 2 O.
- Lanes 1 and 3 which contained no tagged cDNA, gave no amplified products and only the primers were visible.
- Lanes 2 and 4 contained amplified products and in each case, a major product band was observed migrating at the expected molecular weight for the GAPDH (1073 bp) or ⁇ -actin (1151 bp) products respectively, which corresponded to the sequence tag present at the proximal 3' ends of the respective full-length cDNA species.
- Southern blot analysis ( Figure 6 at B and C, Lanes 2 and 4) confirms the amplified products as GAPDH and ⁇ -actin respectively. It is also possible that the ⁇ -actin probe will hybridize to ⁇ -actin sequences, which will be amplified by these primers as well.
- Figure 6 contains the following: Lane 1 - no added template
- reaction #4 A 2- ⁇ L aliquot of the PCR-amplified materials for ⁇ -/ ⁇ -actin as generated in Example 3, reaction #4, was used as template in a secondary PCR reaction containing the first primer (Seq. ID. No. 4) and a gene specific reverse primer for ⁇ -/ ⁇ -actin (Seq. ID. No. 7; AACCCTGCGGCCGCCACATCTGCTGGAAGGTGGACA) now containing a 5' Not I restriction endonuclease site to aid in cloning.
- the PCR reaction was performed as described in Example 3.
- the completed PCR reaction was then purified using the Qia-PCR clean-up procedure (Qiagen) and products corresponding to 50% of the purified reaction was concentrated and separated by agarose gel electrophoresis. A major product band corresponding to actin was then excised and digested with restriction endonucleases Asc I and Not I, in a 50- ⁇ L reaction comprising 20 mM Tris- acetate 9 (pH 7.9), 50 mM KOAc, 1 mM DTT, 100 ⁇ g/mL BSA and 10 units of each enzyme (NEB).
- the digestion reaction was incubated at 37°C for 3 hours, purified using the Qia-PCR clean-up procedure, concentrated into a 2- ⁇ L aliquot and used in a ligation reaction.
- the ligation reaction comprised Asc I-Not I digested PCR amplicons (2 ⁇ L) and 20 ng plasmid vector (pCATRMAN) for cloning in E. coli, 50 mM Tris-HCI (pH 7.5), 10 mM MgCI 2 , 10 mM DTT, 1 mM ATP, 25 ⁇ g/mL BSA and 400 units T4
- DNA ligase (NEB). The ligation reaction was incubated at 16°C overnight, which was followed by 65°C for 10 minutes. To the ligation reaction, 90 ⁇ L H 2 O and 1 mL butanol were added, mixed, and the precipitate collected by centrifugation and reconstituted in 4 ⁇ L H 2 O. A 2- ⁇ L aliquot was then used to transform E. coli (DH10B) by electroporation (Invitrogen).
- NEB DNA ligase
- Table 1 shows a summary of approximately the first 80 nucleotides from the 5' ends of the ⁇ -actin clones that were sequenced to demonstrate the presence of the oligonucleotide sequence tag (shown italicized and bolded).
- RNA from undifferentiated (precursor) and fully differentiated (osteoclast) mouse RAW 264.7 cells was extracted using a Trizol method (InVitrogen), purified further by RNeasy (Qiagen) and quantified at A 260 nm - The precursor and osteoclast specific total RNA samples were then mixed in the following ratios:
- RNA or RNA mixture was then synthesized from each RNA or RNA mixture and first DNA templates prepared using the oligonucleotide sequence tag (Seq. ID. No. 2) according to the teachings of Example 1.
- Each first DNA templates was subsequently annealed to a second oligonucleotide template containing a T7 promoter sequence and a oligonucleotide sequence tag complement to tag sequence contained in the first DNA templates (Seq. ID. No. 3) and an enzymatic DNA polymerization reaction for each performed as described in Example 2.
- RNA 500 ng
- Northern blot hybridization to a 32 P labeled cDNA probe specific for mouse cathepsin K gene.
- Lanes 1-5 show the library of linearly transcribed RNA synthesized from the second DNA templates corresponding to the various RNA and RNA mixtures and in all cases, the profile of the transcribed RNA appear to be similar.
- Figure 7 at B Lanes 1-5 show the Northern blot hybridization results for the cathepsin K gene - Lane 1 , representing the 100% precursor RNA, showed no cathepsin K signal since this is an osteoclast-specific gene and is not expected to be seen in the precursor sample.
- Lanes 2-5 show increasing levels of the cathepsin K gene corresponding to the increasing starting amounts of osteoclast RNA (25% -100%) in each RNA mixture.
- each of the five lanes of the Northern blot was excised and the radioactivity measured by scintillation counting. The counts per minute (cpm) obtained for each of the five lanes, minus the background, was then plotted against the corresponding total RNA or RNA mixtures. As shown in FIG 8, a linear relationship between the increasing levels of osteoclast total RNA in the RNA mixture and the level of cathepsin K signal was observed. This indicates that the tagging procedure does not appear to introduce a bias for this targeted sequence within the total RNA input range tested.
- Lane 2 - 500 ng transcribed RNA from 75% precursor + 25% osteoclast
- Lane 3 - 500 ng transcribed RNA from 50% precursor + 50% osteoclast
- Lane 4 - 500 ng transcribed RNA from 25% precursor + 75% osteoclast
- Lane 5 500 ng transcribed RNA from 100% osteoclast
- the amounts of total RNA purified from the 1000 - 100000 samples were not quantified. Rather, the whole amount of total RNA extracted from each cell dilution was used directly in the tagging and transcription procedures.
- each RNA sample was used for making first-strand cDNA and each cDNA was tagged with the oligonucleotide sequence tag (Seq. ID. No. 2) to generate first DNA templates and purified according to Example 1.
- Each first DNA templates was subsequently annealed to the second oligonucleotide containing a sequence tag complement to the tag contained in the first DNA templates and a T7 promoter sequence (Seq. ID. No. 3), and an enzymatic DNA polymerization reaction for each performed as described in Example 2.
- the resulting second DNA templates containing the double-stranded T7 promoter for each reaction was purified and transcribed in vitro using T7 RNA polymerase as described in Example 2.
- RNA obtained for each total RNA sample after two rounds of transcription is summarized in Table 2.
- Table 2 shows the sensitivity of the terminal tagging procedure comparing purified total RNA diluted from a concentrated stock or purified directly from dilutions of cells.
- the modified selective terminal tagging method was initially compared to the standard method described in the above mentioned EXAMPLES using an input of 100 ng total RNA from fully differentiated mouse RAW 264.7 osteoclasts. The results showed no difference in the yield and quality of the transcribed RNA from both methods (data not shown). Thus, this ensuing example describes the use of a 2 ng input total RNA, in order to further test the sensitivity the modified method compared to the standard method, which is already known to perform well at this level.
- First-strand cDNA was synthesized from two 2 ng samples of total RNA purified from fully differentiated mouse RAW 264.7 osteoclasts according to the teachings of Example 1 with the exception that RNase I was used instead of RNase A. Upon completion of the cDNA synthesis and RNA hydrolysis, one 2 ng reaction was subjected to the standard process for synthesis of first DNA templates and completely double-stranded second DNA templates as described in Example 1 and Example 2.
- the reaction was then incubated at 5O 0 C for 30 minutes.
- RNase H digestion resulted in the removal of at least the 3' terminus blocking nucleotide from the promoter oligonucleotides sequence tag template and thus, converted the promoter sequence tag oligonucleotides template to oligonucleotides primer for directing DNA synthesis using AMV reverse transcriptase.
- the nucleic acids in the reaction mixture was purified using Qiagen MinElute Kit. However, it is contemplated that this purification step of the second DNA templates prior to transcription may not be required.
- the purified tagged second DNA templates from both the 2 ng-standard and 2 ng-modified samples were added to separate 40- ⁇ L in vitro transcription reaction (Ambion) according to the teachings of Example 2 and each transcription reaction was allowed to proceed at 37 0 C for 4 hours.
- the RNA synthesized from each reaction was subjected to a second round of transcription amplification following the teachings of Example 6.
- RNA after the second round was purified (Qiagen Rneasy Kit) and quantified at A 26 o nm - A similar quantity of transcribed RNA was obtained for each sample showing comparable sensitivity.
- a 200 ng of each transcribed RNA (standard and modified methods) was analyzed by agarose gel electrophoresis and Northern blot hybridization to 32 P labeled cDNA probes specific for GAPDH and mouse TRAP genes. Also, 200 ng of the total RNA was compared on the same agarose gel.
- Fig. 12-panel A Lanes 2 and 3 show the electrophoretic profile of the transcribed RNA RNA for the standard method and the modified method respectively. In both cases, the profile of the transcribed RNA appears to be similar.
- Fig. 12-panel B Lanes 2 and 3 show the Northern blot hybridization results for the GAPDH and TRAP genes. Comparing the hybridization pattern in Lane 2 (standard method) and Lane 3 (modified method) with that of total RNA (Lane 1 ), it is evident that the full-length RNA present for both genes was essentially similar for both methods used. These results suggest that the modified method, which is more simplified and homogeneous, and uses 2 instead of 3 oligonucleotides for terminal tagging and efficient RNA synthesis, is a significant improvement to the standard method.
- Fig. 12 contains the following:
- EXAMPLE 8 Selective terminal tagging of cDNA with a promoter containing sequence tag oligonucleotides comprising deoxynucleotides and a blocked 3' terminus followed by RNA synthesis from the single-stranded cDNA template strand
- First-strand cDNA was synthesized from two 50 ng samples of total RNA purified from fully differentiated human osteoclasts according to the teachings of Example 1. Following RNA hydrolysis, one 50 ng cDNA reaction was tagged with Seq. ID. No. 2 following the teachings as described in Examples 1 with the exception that AMV reverse transcriptase in the supplier's reaction buffer (In Vitrogen) was used instead of Klenow's fragment (3' to 5' exo " ). Transcription of the resulting first DNA templates was then performed according to the teachings of Example 2 in order to synthesize amplified RNA.
- AMV reverse transcriptase in the supplier's reaction buffer In Vitrogen
- AATTCTAATACGACTCACTATAGGGAGACGAAGACAGTAGACANNNNNN(N(2'-O- Methyl))(3'-C3 propyl spacer) was added instead of Seq. ID. NO. 2 and the tagging reaction according to the teachings of Example 1 was performed with the exception that AMV reverse transcriptase in the supplier's reaction buffer was used instead of Klenow fragment (3' to 5' exo " ).
- the first DNA templates are formed comprising a functional double-stranded promoter sequence and a single-stranded cDNA template strand since DNA polymerization from the 3' terminus of the sequence tag oligonucleotides template was blocked.
- the first DNA templates comprising the double-stranded promoter was purified using Qiagen MinElute Kit and RNA synthesized directly in an in vitro transcription reaction as described in Example 2 above.
- RNA synthesized from each 50 ng cDNA tagging reactions was then subjected to a second round of transcription amplification following the teachings of Example 6.
- the transcribed RNA after the second round for each reaction was purified (Qiagen Rneasy Kit) and quantified at A 260nm - Table 3 below shows the respective RNA yields from the fully double-stranded second DNA templates formed using SEQ. ID. NO. 2 and single-stranded first DNA templates comprising the double-stranded promoter formed using SEQ. ID. NO. 16.
- SEQ. ID. NO. 16 produced a similar quality transcribed RNA as SEQ. ID. NO. 2 as determined by electrophoresis and hybridization analysis, the yield was approximately 80% lower.
- First-strand cDNA was synthesized from six 50 ng samples of total RNA purified from fully differentiated human osteoclasts according to the teachings of Example 1. After RNA hydrolysis, the following tagging reactions were performed as described in Examples 1 with the exception that AMV reverse transcriptase in the supplier's reaction buffer (In Vitrogen) was used instead of Klenow's fragment (3' to 5' exo ⁇ ): (1) two 50 ng cDNA reaction were tagged with two different synthesis of the oligonucleotide sequence tag corresponding to Seq. ID. No.
- the DNA templates from each of the tagged reactions were used for priming DNA synthesis using a second oligonucleotide template containing a 5' T7 promoter sequence (italicized) and a 3' sequence tag complement corresponding to the respective sequence tag contained in the first DNA templates to form second DNA templates containing a T7 promoter sequence (SEQ ID NO.: No. 3;
- SEQ ID NO.: 17 three different second oligonucleotide templates were tested - SEQ ID NO.: 19 was considered the standard, SEQ ID NO.: 20 was longer by one nucleotide at its 3' end and SEQ ID NO.: 21 was blocked at its 3' terminus with a C3 propyl spacer.
- Transcription of the resulting first DNA templates was then performed according to the teachings of Example 2 of the original patent application in order to synthesize amplified RNA.
- the RNA synthesized from each 50 ng cDNA tagging reactions was then subjected to a second round of transcription amplification following the teachings of Example 6 of the original patent application.
- RNA after the second round for each reaction was purified (Qiagen Rneasy Kit) and the yields quantified at A 260nm (Table 4). Except for the reaction containing the second oligonucleotide template with the blocked 3' terminus (SEQ ID NO.: 21), a similar quantity of transcribed RNA was obtained for each reaction showing comparable sensitivity for the different oligonucleotide sequence tags.
- the incomplete double-stranded second DNA templates formed using SEQ ID NO.: 21 resulted in 80%-90% less transcribed RNA, which was consistent with the findings of Example 2 above using Seq. ID. No. 2. Additionally, two different oligonucleotide synthesis of Seq. ID. No. 2 gave similar yields indicating that the oligonucleotides can be remade without any adverse effects.
- RNA was analyzed by agarose gel electrophoresis and Northern blot hybridization to 32 P labeled cDNA probes specific for GAPDH.
- the electrophoretic profiles of the transcribed RNA for all the different conditions tested were similar and the hybridization pattern for GAPDH indicated largely the full-length product in each case (data not shown).
- AATTCTAATACGACTCACTATAGGGAGACGAAGACAGTAGACArNrN rNrNrNrN(N(2' -O-Methyl))(3'-C3 propyl spacer) rN an equal mixture of ribonucleotides ATP, GTP, CTP and UTP.
- Oligonucleotide Sequence Tag (SEQ ID NO.: 18): GCCTGCACCAACAGTTCACAGANNNNNN (N-2Omethyl)-3'-C3 propyl spacer
- Second Oligonucleotide Template (SEQ ID NO.: 19): AA TTCTAA TA CG ACTCACTA TAGGGAGAGCCTGCACCAACAGTTAAC
- Second Oligonucleotide Template (SEQ ID NO.: 20): AATTCTAATACGACTCACTATAGGGAGAGCCTGCACCAACAGJTAACA
- Second Oligonucleotide Template (SEQ ID NO.:22): AATTCTAATACGACTCACTATAGGGAGAGCCTGCACCAACAGTTCACA
Abstract
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PCT/CA2005/001830 WO2007062495A1 (en) | 2005-11-30 | 2005-11-30 | Selective terminal tagging of nucleic acids |
US12/095,409 US8304183B2 (en) | 2005-11-30 | 2005-11-30 | Selective terminal tagging of nucleic acids |
CA2639819A CA2639819C (en) | 2005-11-30 | 2005-11-30 | Selective terminal tagging of nucleic acids |
EP05814417A EP1954706B1 (en) | 2005-11-30 | 2005-11-30 | Method using reversibly blocked tagging oligonucleotides |
AT05814417T ATE551349T1 (en) | 2005-11-30 | 2005-11-30 | METHOD USING REVERSIBLY BLOCKED LABELING OLIGONUCLEOTIDES |
AU2005338632A AU2005338632B2 (en) | 2005-11-30 | 2005-11-30 | Selective terminal tagging of nucleic acids |
US13/331,533 US20120156729A1 (en) | 2003-12-02 | 2011-12-20 | Selective terminal tagging of nucleic acids |
US13/627,616 US20130029881A1 (en) | 2005-11-30 | 2012-09-26 | Selective terminal tagging of nucleic acids |
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Also Published As
Publication number | Publication date |
---|---|
CA2639819A1 (en) | 2007-06-07 |
EP1954706A4 (en) | 2009-11-18 |
US20090227009A1 (en) | 2009-09-10 |
US8304183B2 (en) | 2012-11-06 |
US20130029881A1 (en) | 2013-01-31 |
EP1954706B1 (en) | 2012-03-28 |
AU2005338632B2 (en) | 2010-05-20 |
CA2639819C (en) | 2012-10-23 |
EP1954706A1 (en) | 2008-08-13 |
AU2005338632A1 (en) | 2007-06-07 |
ATE551349T1 (en) | 2012-04-15 |
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