Amplification of nucleic acid sequences is a widespread technology that has been used for many purposes, including diagnostic and forensic testing. Currently, this is carried out using polymerase chain reaction (PCR). Unfortunately, critical barriers exist with PCR that prevent both clinical and research labs from adopting PCR-based assays into a routine setting, due to bottlenecks with sample preparation and assay development costs. Specifically, the PCR inhibitors, such as inhibitors to polymerases, found in many laboratory samples and clinical specimens cause low sensitivity and false-negative results in clinical and forensic tests that rely on PCR-based molecular techniques. Therefore, it is widely accepted that purification or pre-amplification of target DNA nucleic acids is required to remove or dilute out inhibitors prior to PCR amplification to obtain successful results. Optimization of PCR for genetic testing with different sample types can be labor intensive, requiring extensive amounts of upfront development work, which in turn can significantly increase both the overall cost of a test and the time-to-result.1-5 With the upsurge in genetic information and the resultant increase in DNA biomarkers, researchers are now seeking new technologies to rapidly and cost-effectively interrogate this new information in a routine setting. However, the critical barriers associated with PCR make this technology too cost-prohibitive and too labor-intensive to use as a testing method for price-sensitive laboratories with limited resources and large numbers of samples.
Recently, technology has been developed to detect and monitor cellular genetic mutations using RNA-templated chemistry, in which chemically modified probes fluoresce when they hybridize to their genetic target in intact bacterial and human cells.10-15 This probe-based strategy, called quenched autoligation (QUAL), utilizes two self-reacting oligonucleotide probes that provide a fluorescence signal in the presence of fully complementary nucleic acid target sequence. A first oligonucleotide having a 3′-phosphoromono-thioate nucleophilic group anneals to a template target sequence, such that the 3′-phosphoromono-thioate nucleophilic group is juxtaposed to a 5′-electrophilic dabsylated group quencher of a second annealed oligonucleotide which has a fluorescein group quenched by the dabsyl group. This tandem configuration along a DNA template catalyzes the autoligation reaction, and joins the two oligonucleotides into a single probe. Upon ligation, the dabsyl quencher is displaced, and the fluoresceinyl fluorophore becomes un-quenched, resulting in an increase in fluorescence signal.
These short QUAL probes have been used to distinguish closely related bacterial species by discriminating single nucleotide differences in 16S rRNA sequences within live cells. QUAL offers the potential to develop new bioanalytical assays in living cells, such as RNA localization, transcription, and RNA processing. However, this strategy is not compatible with in vitro applications that require the detection of small amounts of double-stranded nucleic acid sequences that are typically found in samples used for routine genetic testing of DNA biomarkers. For example, a QUAL in vitro reaction typically contains 1013 copies of single-stranded oligo DNA template, but a routine molecular assay can contain 103 or fewer copies of dsDNA biomarkers—a ten billion-fold difference in copy-number detection. Detecting such a small number of molecules requires amplification of those numbers, which in turn requires thermal denaturation of the double stranded DNA molecules. Unfortunately, the autoligation chemistries used in QUAL are not thermostable enough to last more than a few minutes at the high temperatures needed to separate double-stranded DNA. Thus, these procedures are not suitable for amplifying nucleic acid sequences.
There is, therefore, a need for thermostable reagents and methods for amplifying nucleic acid sequences without enzymes or nucleosides to enable cost-effective and easier-to-use alternatives for genetic testing that can be implemented in routine settings across multiple sample types without any sample-prep development.
The invention relates to amplification of nucleic acid sequences. More particularly, the invention relates to amplification of nucleic acid sequences without enzymes or nucleosides. The invention provides thermostable reagents and methods for amplifying nucleic acid sequences without enzymes or nucleosides. In a first aspect, the invention provides a method for exponentially amplifying a specific target nucleic acid sequence. The method according to this aspect of the invention comprises contacting the target nucleic acid sequence with a first forward primer nucleic acid, a second forward primer nucleic acid, a first reverse primer nucleic acid and a second reverse primer nucleic acid under conditions wherein the primer nucleic acids specifically anneal with the target nucleic acid sequence. One forward primer nucleic acid has a thermally stable first bond-forming reactive moiety and the other forward primer nucleic acid has a thermally stable second bond-forming reactive moiety. One reverse primer nucleic acid has a thermally stable first bond-forming reactive moiety and the other reverse primer nucleic acid has a thermally stable second bond-forming reactive moiety. The first forward primer nucleic acid and the second forward primer nucleic acid are annealed to the target nucleic acid sequence such that the reactive moiety of the first forward primer nucleic acid and the reactive moiety of the second forward primer nucleic acid are juxtaposed. The first reverse primer nucleic acid and the second reverse primer nucleic acid are annealed to the target nucleic acid sequence such that the reactive moiety of the first reverse primer nucleic acid and the reactive moiety of the second reverse primer nucleic acid are juxtaposed. The reactive moiety of the first forward primer nucleic acid forms a chemical bond with the reactive moiety of the second forward primer nucleic acid to form a first ligation product, and the reactive moiety of the first reverse primer nucleic acid forms a chemical bond with the reactive moiety of the second reverse primer nucleic acid to form a second ligation product. Thus, the first ligation product forms a duplex with the target nucleic acid sequence and the second ligation product forms a duplex with the target nucleic acid sequence. The duplexes are then thermally disrupted to form target nucleic acid sequences and the steps are repeated to exponentially amplify the target nucleic acid sequences.
In some embodiments, the thermally stable first bond-forming reactive moiety is a nucleophilic moiety and the thermally stable second bond-forming reactive moiety is an electrophilic moiety. In some embodiments, the thermally stable first bond-forming reactive moiety is an electrophilic moiety and the thermally stable second bond-forming reactive moiety is a nucleophilic moiety.
In some embodiments, one forward or reverse primer nucleic acid comprises a dye or detectable group. In some embodiments, one forward or reverse primer nucleic acid comprises a fluorescence resonance energy transfer (FRET) donor fluorophore and/or the other forward or reverse primer nucleic acid comprises a FRET acceptor fluorophore, and the ligation products are detected by FRET.
In a second aspect, the invention provides reagents for exponentially amplifying a target nucleic acid sequence. In some embodiments, a reagent according to the invention comprises a first forward primer nucleic acid having a thermally stable first bond-forming reactive moiety. In some embodiments, a reagent according to the invention comprises a second forward primer nucleic acid having a thermally stable second bond-forming reactive moiety. In some embodiments, a reagent according to the invention comprises a first reverse primer nucleic acid having a thermally stable first bond-forming reactive moiety. In some embodiments, a reagent according to the invention comprises a second reverse primer nucleic acid having a thermally stable second bond-forming reactive moiety. In such embodiments, the first bond-forming reactive moiety forms a chemical bond with the second bond-forming reactive moiety, when the first forward primer nucleic acid and the second forward primer nucleic acid are juxtaposed by annealing with a target nucleic acid and when the first reverse primer and the second reverse primer nucleic acid are juxtaposed by annealing with a target nucleic acid. In some embodiments, the first bond-forming reactive moiety is a nucleophilic moiety and the second bond-forming reactive moiety is an electrophilic moiety. In some embodiments, the first bond-forming reactive moiety is an electrophilic moiety and the second bond-forming reactive moiety is a nucleophilic moiety. In some embodiments, one forward or reverse primer nucleic acid comprises a dye or detectable group. In some embodiments, one forward or reverse primer nucleic acid comprises a FRET donor fluorophore and/or the other forward or reverse primer nucleic acid comprises a FRET acceptor fluorophore.
In a third aspect, the invention provides a kit for exponentially amplifying a target nucleic acid sequence. The kit according to this aspect of the invention comprises a first forward primer nucleic acid, a second forward primer nucleic acid, a first reverse primer nucleic acid, and a second reverse primer nucleic acid. In the kit according to this aspect of the invention, the first forward primer nucleic acid, the second forward primer nucleic acid, the first reverse primer nucleic acid, and the second reverse primer nucleic acid are as described for the second aspect according to the invention.
The foregoing will be apparent from the following more particular description of example embodiments of the invention, as illustrated in the accompanying drawings.
A description of example embodiments of the invention follows.
The invention relates to amplification of nucleic acid sequences. More particularly, the invention relates to amplification of nucleic acid sequences without enzymes or nucleosides. The invention provides thermostable reagents and methods for amplifying nucleic acid sequences without enzymes or nucleosides.
In a first aspect, the invention provides a method for exponentially amplifying a specific target nucleic acid sequence. The method according to this aspect of the invention comprises contacting the target nucleic acid sequence with a first forward primer nucleic acid, a second forward primer nucleic acid, a first reverse primer nucleic acid and a second reverse primer nucleic acid under conditions wherein the primer nucleic acids specifically anneal with the target nucleic acid sequence. One forward primer nucleic acid has a thermally stable first bond-forming reactive moiety and the other forward primer nucleic acid has a thermally stable second bond-forming reactive moiety. One reverse primer nucleic acid has a thermally stable first bond-forming reactive moiety and the other reverse primer nucleic acid has a thermally stable second bond-forming reactive moiety. The first forward primer nucleic acid and the second forward primer nucleic acid are annealed to the target nucleic acid sequence such that the reactive moiety of the first forward primer nucleic acid and the reactive moiety of the second forward primer nucleic acid are juxtaposed. The first reverse primer nucleic acid and the second reverse primer nucleic acid are annealed to the target nucleic acid sequence such that the reactive moiety of the first reverse primer nucleic acid and the reactive moiety of the second reverse primer nucleic acid are juxtaposed. The reactive moiety of the first forward primer nucleic acid forms a chemical bond with the reactive moiety of the second forward primer nucleic acid to form a first ligation product, and the reactive moiety of the first reverse primer nucleic acid forms a chemical bond with the reactive moiety of the second reverse primer nucleic acid to form a second ligation product. Thus, the first ligation product forms a duplex with the target nucleic acid sequence and the second ligation product forms a duplex with the target nucleic acid sequence. The duplexes are then thermally disrupted to form target nucleic acid sequences and the steps are repeated to exponentially amplify the target nucleic acid sequences.
In some embodiments, the thermally stable first bond-forming reactive moiety is a nucleophilic moiety and the thermally stable second bond-forming reactive moiety is an electrophilic moiety. In some embodiments, the thermally stable first bond-forming reactive moiety is an electrophilic moiety and the thermally stable second bond-forming reactive moiety is a nucleophilic moiety.
In some embodiments, one forward or reverse primer nucleic acid comprises a dye or detectable group. In some embodiments, one forward or reverse primer nucleic acid comprises a FRET donor fluorophore and/or the other forward or reverse primer nucleic acid comprises a FRET acceptor fluorophore, and the ligation products are detected by FRET.
In a second aspect, the invention provides reagents for exponentially amplifying a target nucleic acid sequence. In some embodiments, a reagent according to the invention comprises a first forward primer nucleic acid having a thermally stable first bond-forming reactive moiety. In some embodiments, a reagent according to the invention comprises a second forward primer nucleic acid having a thermally stable second bond-forming reactive moiety. In some embodiments, a reagent according to the invention comprises a first reverse primer nucleic acid having a thermally stable first bond-forming reactive moiety. In some embodiments, a reagent according to the invention comprises a second reverse primer nucleic acid having a thermally stable second bond-forming reactive moiety. In such embodiments, the first bond-forming reactive moiety forms a chemical bond with the second bond-forming reactive moiety, when the first forward primer nucleic acid and the second forward primer nucleic acid are juxtaposed by annealing with a target nucleic acid and when the first reverse primer and the second reverse primer nucleic acid are juxtaposed by annealing with a target nucleic acid. In some embodiments, the first bond-forming reactive moiety is a nucleophilic moiety and the second bond-forming reactive moiety is an electrophilic moiety. In some embodiments, the first bond-forming reactive moiety is an electrophilic moiety and the second bond-forming reactive moiety is a nucleophilic moiety. In some embodiments, one forward or reverse primer nucleic acid comprises a dye or detectable group. In some embodiments, one forward or reverse primer nucleic acid comprises a FRET donor fluorophore and/or the other forward or reverse primer nucleic acid comprises a FRET acceptor fluorophore. In a third aspect, the invention provides a kit for exponentially amplifying a target nucleic acid sequence. The kit according to this aspect of the invention comprises a first forward primer nucleic acid, a second forward primer nucleic acid, a first reverse primer nucleic acid, and a second reverse primer nucleic acid. In the kit according to this aspect of the invention, the first forward primer nucleic acid, the second forward primer nucleic acid, the first reverse primer nucleic acid, and the second reverse primer nucleic acid are as described for the second aspect according to the invention.
Non-limiting examples of reagents and methods according to the invention are shown in
Forward ACR Primer 1 and Reverse ACR Primer 2 and Forward ACR Primer 2 and Reverse ACR Primer 1 are complementary pairs, which increase the specificity of the reaction by sequestering the primers in duplexes until dsDNA templates outcompete the formation of oligo homoduplexes by annealing to the oligos. Because tandemly-annealed oligos on a template have significantly higher melting temperatures than individual oligos annealed to the same template, due to stabilizing base-pair stacking interactions between the tandemly-aligned oligos, ACR is performed at annealing temperatures that favor the formation of oligo/template heteroduplexes over homoduplexed oligo sets. For purposes of the invention, a “primer nucleic acid” is an oligonucleotide used in the method according to the invention to form a longer oligonucleotide via autoligation to another primer nucleic acid. Primer nucleic acids may be from about 5 to about 35 nucleotides in length. The autoligation reaction occurs when the primer nucleic acids are annealed to a target nucleic acid sequence such that a first bond-forming reactive moiety of one primer nucleic acid is juxtaposed with a second bond-forming reactive moiety of another primer nucleic acid. In some embodiments the first bond-forming reactive moiety is at a terminus (5′ or 3′) of one primer nucleic acid and the second bond-forming reactive moiety is at an opposite terminus of the other primer nucleic acid. The terms “first bond-forming reactive moiety” and “second bond-forming reactive moiety” refer to chemical functional groups that are capable of reacting with each other to form a covalent bond.
Non-limiting examples of first bond-forming reactive moieties include phosphorodithioate, phosphorotrithioate, 2′,3′-cyclic phosphate, amino-deoxyribonucleosides, thiol, amino, hydrazine and hydrazide. In certain embodiments, the first bond-forming reactive moiety is a nucleophile. A 3′-thionucleoside is a particularly preferred 3′ terminal nucleophile.
Non-limiting examples of second bond-forming reactive moieties include bromide, iodide, chloride, maleimide, dabsylate, pyridyldisulfide, tosylate, alkyne, isothiocyanate, cyclooctyne, NHS ester, imidoester, PFP ester, alkyl azide, aryl azide, isocyanate, nitrophenyl mono- or di-ester and epoxy. In certain embodiments, the second bond-forming reactive moiety is an electrophile. A 5′-bromoacetylnucleoside is a particularly preferred 5′-terminal electrophile.
Amplification of a double-stranded target nucleic acid sequence requires thermal denaturation of the target sequence. Thus, the first and second bond-forming reactive moieties must be thermally stable. “Thermally stable” means that the moiety reactivity must not be destroyed or functionally compromised at temperatures required to denature the target sequence.
In some embodiments, a dye or detectable group is used to detect the ligated products formed by annealing and autoligation. Non-limiting dyes and detectable groups include, without limitation, the groups shown in Table I below.
In some embodiments, the first forward primer and second forward primer or the first reverse primer are conjugated to dyes that are, respectively, a donor dye and an acceptor dye for fluorescence resonance energy transfer (FRET). Alternatively, the first forward primer and second forward primer or the first reverse primer are conjugated to dyes that are, respectively, an acceptor dye and a donor dye for FRET. Alternatively, the donor and acceptor dyes for FRET may be, respectively, on the second reverse primer and the first reverse primer or the second forward primer. Alternatively, the second reverse primer and the first reverse primer or the second forward primer are conjugated to dyes that are, respectively, an acceptor dye and a donor dye for FRET. Alternatively, the first forward primer and second forward primer are conjugated to dyes that are, respectively, an acceptor dye and a donor dye, and the second reverse primer and the first reverse primer are conjugated to dyes that are, respectively, an acceptor dye and a donor dye for FRET. Alternatively, the first forward primer and second forward primer are conjugated to dyes that are, respectively, a donor dye and an acceptor dye, and the second reverse primer and the first reverse primer are conjugated to dyes that are, respectively, a donor dye and an acceptor dye for FRET. In some embodiments, the donor and acceptor dyes are spaced from about 5 to about 10 nucleotides apart within the autoligation product. In a particularly preferred embodiment, the donor dye is FAM and the acceptor dye is Texas Red.
In some embodiments, the dye or detectable group is quenched by a quenching moiety in which annealing and autoligation separates the quenching moiety from the dye or detectable group before the ligated product is detected.
The following examples are intended to further illustrate certain embodiments of the invention and are not to be construed to limit the scope of the invention.
For the initial ACR chemistry we chose a system based on oligonucleotides modified with two reactive chemical groups, a thiol group incorporated at the 3′ end (i.e. nucleophile) and alkyl-halide or maleimide groups incorporated at the 5′ end (i.e. electrophile). We reasoned that this type of chemistry would allow for the nucleophile and electrophile oligos to react with each other only when juxtaposed in close vicinity by hybridizing to the complementary template.9 The first step in the nucleophile primer synthesis involved the preparation of a 3′-thio-2′,3′-dideoxynucleoside building block in the protected form, attached to the custom solid support via disulfide bond with a modification to include a non-standard amino-modifier residue (2′-amino-dT) that carries a short alkyl linker with no double bonds. (
A Tx-Red labeled oligonucleotide (analogous to forward primer 1, shown in
Reactions were performed using unlabeled Forward ACR Primer 1 nucleophile (GCAACGACCGTTCCGT-SH) and labeled Forward ACR Primer 2 electrophile (BrAc-TCAAT(FAM)ACTGCGCAGCC). Increasing ssDNA oligo template was added to reactions in a molar excess. Reactions were set up at room temperature and incubated at 35° C. for 20 min. Reactions were stopped with equal volumes of formamide+dye, heat denatured, cooled on ice, and load directly onto a 20% acrylamide+urea denaturing gel. The reactions worked the best at pH 7 in the presence of 20 mM DTT, at temperatures between 20-40 degrees. The most efficient autoligation was observed with Br-acetate-based electrophiles.
Reactions were performed using unlabeled Reverse ACR Primer 1 nucleophile (GGCTGCGCAGTAT-SH) and unlabeled Reverse ACR Primer 2 electrophile (BrAc-TGAACGGAACGGTCGTTGC). Increasing ssDNA oligo template was added to reactions in a molar excess (lanes 2-5). Lane 1 of each panel is the no-template control. Reactions were set up at room temperature and incubated at 35° C. for 20 min. Reactions were stopped with equal volumes of formamide+dye, heat denatured, cooled on ice, and load directly onto a 20% acrylamide+urea denaturing gel.
Reactions were performed using unlabeled Forward ACR Primer 1 and FAM-labeled Forward ACR Primer 2. ssDNA oligo template was added to the reactions at a 33-fold molar excess. Reactions were set up at room temperature and incubated at 35° C. for 20 min., and then thermocycled in a MultiGene Labnet thermocycler. The thermocycling protocol was 95° C. for 5 min., then 40 cycles of 95° C., 30 sec. and 20° C., 1 min. The reactions were stopped with equal volumes of formamide containing dye, heat denatured, cooled on ice, and load directly onto the denaturing gel.
Reactions were performed using ACR primers with optimized nucleophilic and electrophilic moieties using new thiol/bromoacetate chemistry according to the invention (Lanes 1 and 2), and oligo pairs previously tested with phosphoromono-thioate ester nucleophilic and dabsylate electrophilic chemistries (Lanes 3 and 4). Reactions were thermo-cycled without any enzyme or nucleotides, either in the presence (Lanes 1 and 3) or absence (Lanes 2 and 4) of complementary ssDNA oligonucleotide template.
Reactions were performed using Texas Red-labeled Forward ACR Primer 1, FAM-labeled Forward ACR Primer 2, and unlabeled reverse primers, with ssDNA oligo as template. Reactions were set up on ice and thermocycled for 40 cycles. The reactions were stopped with equal volumes of formamide containing dye, heat denatured, cooled on ice, and loaded directly onto a denaturing gel.
Reactions were performed using Texas Red-labeled Forward ACR Primer 1, FAM-labeled Forward ACR Primer 2, and unlabeled reverse primers, with ssDNA oligo as template. Template was added in 4-fold molar excess over the ACR primers in the reaction. Reactions were set up on ice and thermocycled for 40 cycles. The normalized baselined data was exported into Excel, and the plots were smoothed by a 4-point rolling average of the data.
Reactions were performed using Texas Red-labeled Forward ACR Primer 1, FAM-labeled Forward ACR Primer 2, and unlabeled reverse primers, with a titration of dsDNA oligo template. The reactions were stopped with equal volumes of formamide containing dye, heat denatured, cooled on ice, and load directly onto the denaturing gel.
The teachings of all patents, published applications and references cited herein are incorporated by reference in their entirety.
While this invention has been particularly shown and described with references to example embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.
This application claims the benefit of U.S. Provisional Application No. 61/580,998, filed on Dec. 28, 2011. The entire teachings of the above application are incorporated herein by reference.
This invention was made in part with government support under grant #1046508 awarded to the inventors by the National Science Foundation. The government has certain rights in the invention.
Number | Date | Country | |
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61580998 | Dec 2011 | US |