Patent Application
20100209932
The present teachings are in the field of molecular and cell biology, specifically in the field of multiplexed detection of messenger RNAs along with short nucleic acids such as micro RNAs.
Numerous fields in molecular biology require the identification of target polynucleotide sequences. Reverse transcription and amplification are two frequently used procedures employed to query the identity of target polynucleotides. The increasing amount of sequence information available to scientists in the post-genomics era has produced an increased need for rapid, reliable, low-cost, high-throughput, sensitive, and accurate methods to query complex nucleic acid samples. Methods of defining and characterizing cells have been hindered by robust amplification technologies, as well as the molecular complexity of conventionally analyzed molecules such as messenger RNA. Micro RNAs are a recently discovered class of molecules that offer great promise in understanding cell function. However, quantitative analysis of micro RNA has been hindered by their relatively short size.
The present teachings provide a method of detecting a plurality of short target nucleic acids, and a plurality of messenger RNAs on a same solid support, wherein each short target nucleic acid is 18-25 nucleotides in length, said method comprising; contacting the plurality of different short target nucleic acids with a plurality of target-specific stem-loop reverse transcription primers, wherein each of the plurality of stem-loop reverse transcription primers comprises a 3′ target-specific portion, a zip-code stem, and a promoter; contacting, in the same reaction mixture as the short target nucleic acids, a plurality of messenger RNAs with an oligo-dT-promoter-containing reverse transcription primer; extending the plurality of reverse transcription primers and the oligo-dT-promoter-containing reverse transcription primer in a reverse transcription reaction to form a collection of reverse transcription products; amplifying the reverse transcription products in an in vitro transcription reaction comprising an enzyme corresponding to the promoter, to form a plurality of in vitro transcription products; and, detecting the plurality of in vitro transcription products on the same solid support. Reaction compositions, kits, and devices are also provided.
The skilled artisan will understand that the drawings, described below, are for illustration purposes only. The drawings are not intended to limit the scope of the present teachings in any way.
Aspects of the present teachings may be further understood in light of the following examples, which should not be construed as limiting the scope of the present teachings in any way. The section headings used herein are for organizational purposes only and are not to be construed as limiting the described subject matter in any way. All literature and similar materials cited in this application, including but not limited to, patents, patent applications, articles, books, treatises, and internet web pages are expressly incorporated by reference in their entirety for any purpose. When definitions of terms in incorporated references appear to differ from the definitions provided in the present teachings, the definition provided in the present teachings shall control. It will be appreciated that there is an implied “about” prior to the temperatures, concentrations, times, etc discussed in the present teachings, such that slight and insubstantial deviations are within the scope of the present teachings herein. In this application, the use of the singular includes the plural unless specifically stated otherwise. For example, “a target-specific stem-loop reverse transcription primer” means that more than one target-specific stem-loop reverse transcription primer can, but need not be present; for example but without limitation, one or more copies of a particular primer species, as well as one or more versions of a particular primer type. Also, the use of “comprise”, “comprises”, “comprising”, “contain”, “contains”, “containing”, “include”, “includes”, and “including” are not intended to be limiting. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention.
As used herein, the term “short target nucleic acid” refers to a polynucleotide sequence that is sought to be amplified and quantitated, and that is between 18 and 25 nucleotides in length. The short target nucleic can be obtained from any source, and can comprise any number of different compositional components. For example, the target nucleic acid can be DNA, RNA, transfer RNA, siRNA, and can comprise nucleic acid analogs or other nucleic acid mimics, though typically the short target nucleic acids will be micro RNAs (miRNAs) and other short RNAs. The target can be methylated, non-methylated, or both. The target can be bisulfite-treated and non-methylated cytosines converted to uracil. Further, it will be appreciated that “short target nucleic acid” can refer to the short target nucleic acid itself, as well as surrogates thereof, for example amplification products, and native sequences. In some embodiments, the short target nucleic is a short DNA molecule derived from a degraded source, such as can be found in for example but not limited to forensics samples (see for example Butler, 2001, Forensic DNA Typing: Biology and Technology Behind STR Markers. The short target nucleic acid of the present teachings can be derived from any of a number of sources, including without limitation, viruses, prokaryotes, eukaryotes, for example but not limited to plants, fungi, and animals. These sources may include, but are not limited to, whole blood, a tissue biopsy, lymph, bone marrow, amniotic fluid, hair, skin, semen, biowarfare agents, anal secretions, vaginal secretions, perspiration, saliva, buccal swabs, various environmental samples (for example, agricultural, water, and soil), research samples generally, purified samples generally, cultured cells, and lysed cells. It will be appreciated that short target nucleic acids can be isolated from samples using any of a variety of procedures known in the art, for example the Applied Biosystems ABI Prism™ 6100 Nucleic Acid PrepStation, and the ABI Prism™ 6700 Automated Nucleic Acid Workstation, Boom et al., U.S. Pat. No. 5,234,809, mirVana RNA isolation kit (Ambion), etc. It will be appreciated that polynucleotides can be cut or sheared prior to analysis, including the use of such procedures as mechanical force, sonication, restriction endonuclease cleavage, or any method known in the art, to produce short target nucleic acids. In general, the short target nucleic acids of the present teachings will be single stranded, though in some embodiments the short target nucleic can be double stranded, and/or comprise double-stranded regions due to secondary structure, and a single strand can result from denaturation
As used herein, the term “reverse transcription reaction” refers to an elongation reaction in which the 3′ target-specific portion of a target-specific stem-loop reverse transcription primer, and the oligo-dT end of the oligo-dT-promoter-containing reverse transcription primer, are extended to form an extension reaction product comprising complementary strands to the short target nucleic acids and the messenger RNAs. In some embodiments, the short target nucleic acid is a miRNA molecule and the extension reaction is a reverse transcription reaction comprising a reverse transcriptase, where the 3′ end of a target-specific stem-loop reverse transcription primer is extended. In some embodiments, the extension reaction is a reverse transcription reaction comprising a polymerase derived from a Eubacteria. In some embodiments, the extension reaction can comprise rTth polymerase, for example as commercially available from Applied Biosystems catalog number N808-0192, and N808-0098.
In some embodiments, the short target nucleic acid is a DNA molecule and the extension reaction comprises a polymerase and results in the synthesis of a complementary strand of DNA. The term reverse transcription can thus also include the synthesis of a DNA complement of a template DNA molecule, as well as the synthesis of a DNA complement of a template RNA molecule.
As used herein, the term “hybridization” refers to the complementary base-pairing interaction of one nucleic acid with another nucleic acid that results in the formation of a duplex, triplex, or other higher-ordered structure, and is used herein interchangeably with “annealing.” Typically, the primary interaction is base specific, e.g., A/T and G/C, by Watson/Crick and Hoogsteen-type hydrogen bonding. Base-stacking and hydrophobic interactions can also contribute to duplex stability. Conditions for hybridizing primers to complementary and substantially complementary target sequences are well known, e.g., as described in Nucleic Acid Hybridization, A Practical Approach, B. Hames and S. Higgins, eds., IRL Press, Washington, D.C. (1985) and J. Wetmur and N. Davidson, Mol. Biol. 31:349 et seq. (1968). In general, whether such annealing takes place is influenced by, among other things, the length of the polynucleotides and the complementary, the pH, the temperature, the presence of mono- and divalent cations, the proportion of G and C nucleotides in the hybridizing region, the viscosity of the medium, and the presence of denaturants. Such variables influence the time required for hybridization. Thus, the preferred annealing conditions will depend upon the particular application. Such conditions, however, can be routinely determined by the person of ordinary skill in the art without undue experimentation. It will be appreciated that complementarity need not be perfect; there can be a small number of base pair mismatches that will minimally interfere with hybridization between the target sequence and the primers of the present teachings. However, if the number of base pair mismatches is so great that no hybridization can occur under minimally stringent conditions then the sequence is generally not a complementary target sequence. Thus, complementarity herein is meant that primers are sufficiently complementary to the target sequence to hybridize under the selected reaction conditions to achieve the ends of the present teachings. Likewise, the immobilized probes on the solid support are sufficiently complementary to the in vitro transcription products to hybridize under the selected reaction conditions to achieve the ends of the present teachings.
The term “corresponding” as used herein refers to a specific relationship between the elements to which the term refers. Some non-limiting examples of corresponding include: a stem-loop reverse transcription primer can correspond to a particular short target nucleic acid, the in vitro transcription product of a particular species of short target nucleic acid can correspond to a particular immobilized probe on a solid support, etc.
As used herein, the term “detection” refers to the application of any of a variety of microarrays, labeling procedures, and related software such as the Applied Biosystems Array System with the Applied Biosystems 1700 Chemiluminescent Microarray Analyzer and other commercially available array systems available from Affymetrix, Agilent, Illumina, and Amersham Biosciences, among others (see also Gerry et al., J. Mol. Biol. 292:251-62, 1999; De Bellis et al., Minerva Biotec 14:247-52, 2002; and Stears et al., Nat. Med. 9:140-45, including supplements, 2003). It will also be appreciated that detection can comprise reporter groups that are incorporated into the reaction products, for example due to the incorporation of labeled dNTPs during an in vitro amplification, or attached to reaction products, for example but not limited to the inclusion DIG-labeled dUTP (Digoxigenin-labeled dUTP) in the reaction, with subsequent labeling with alkaline-phosphatase-based chemiluminescence. Some illustrative detection methods are further described in U.S. Pat. No. 6,905,826.
As used herein, the term “target-specific stem-loop reverse transcription primer” refers to a molecule comprising a 3′ target specific portion, a stem, and a loop. Illustrative target-specific stem-loop reverse transcription primers are depicted in
As used herein, the term “zip-code stem” refers to the double-stranded region of the target-specific stem-loop reverse transcription primer. The stem of the target-specific stem-loop reverse transcription primer can be 8 base-pairs in length. In some embodiments, the stem can be 9 base-pairs in length. In some embodiments, the stem can be 10 base-pairs in length. In some embodiments, the stem can be 11 base-pairs in length. In some embodiments, the stem can be 12 base-pairs in length. In some embodiments, the stem can be 13 base-pairs in length. In some embodiments, the stem can be 14 base-pairs in length. Generally, longer stems are possible, but will come at the cost of increased expense in oligonucleotide manufacturing, and will further add to reaction complexity. In some embodiments, the stem can be 7 base-pairs in length. In some embodiments, the stem can be 6 base-pairs in length. Stems shorter than 6 base-pairs in length are possible, but are done at the sacrifice of specificity at the level of binding to the immobilized probe on an array. Generally, one of the complementary strands of the stem can comprise an identifying portion, referred to herein as a “zip-code.” Descriptions of zip-codes can be found in, among other places, U.S. Pat. Nos. 6,309,829 (referred to as “tag segment” therein); 6,451,525 (referred to as “tag segment” therein); 6,309,829 (referred to as “tag segment” therein); 5,981,176 (referred to as “grid oligonucleotides” therein); 5,935,793 (referred to as “identifier tags” therein); and PCT Publication No. WO 01/92579 (referred to as “addressable support-specific sequences” therein).
As used herein, one can distinguish between the two complementary strands of the stem by the terms “3′ stem region of the target-specific stem-loop reverse transcription primer,” which refers to the strand nearest to the 3′ end of the stem-loop reverse transcription primer. The other stand can be referred to as the “5′ stem region of the stem-loop reverse transcription primer.”
Thus, in some embodiments the present teachings provide a method of detecting a plurality of short target nucleic acids, and a plurality of messenger RNAs on a same solid support, wherein each short target nucleic acid is 18-25 nucleotides in length, said method comprising; contacting the plurality of different short target nucleic acids with a plurality of target-specific stem-loop reverse transcription primers, wherein each of the plurality of stem-loop reverse transcription primers comprises a 3′ target-specific portion, a zip-code stem, and a promoter; contacting, in the same reaction mixture as the short target nucleic acids, a plurality of messenger RNAs with an oligo-dT-promoter-containing reverse transcription primer; extending the plurality of target-specific reverse transcription primers and the oligo-dT-promoter-containing reverse transcription primer in a reverse transcription reaction to form a collection of reverse transcription products; amplifying the reverse transcription products in an in vitro transcription reaction comprising an enzyme corresponding to the promoter, to form a plurality of in vitro transcription products; and, detecting the plurality of in vitro transcription products on the same solid support. In some embodiments, the plurality of short target nucleic acids are micro RNAs. In some embodiments, the loop of the target-specific stem-loop reverse transcription primers comprises the promoter. In some embodiments, the promoter is T7, though any of a variety of other promoters may be employed, including for example SP6 and T3. Methods employing promoter sequences to effectuate in vitro transcription are known, and can be found for example in U.S. Pat. No. 5,514,545, U.S. Pat. No. 5,545,522, U.S. Pat. No. 5,554,552, U.S. Pat. No. 5,716,785, U.S. Pat. No. 5,891,636, and U.S. Pat. No. 6,114,152. In some embodiments, the in vitro transcription reaction comprises DIG-dUTP, though any of a variety of labeling means can be employed, including the amino-allyl labeling procedure. In some embodiments, the plurality of short target nucleic acids comprises at least 100 different short target nucleic acid species. In some embodiments, the plurality of short target nucleic acids comprises at least 200 different short target nucleic acid species. In some embodiments, the plurality of short target nucleic acids comprises at least 300 different short target nucleic acid species.
In some embodiments, the present teachings provide a reaction composition comprising;
(A) a plurality of target-specific stem-loop reverse transcription primers, wherein each of the plurality of target-specific stem-loop reverse transcription primers comprises a 3′ target-specific portion, a zip-code stem, a promoter; and,
(B) an oligo-dT-promoter-containing reverse transcription primer. In some embodiments, the plurality of target-specific stem-loop reverse transcription primers comprise at least 100 target-specific stem-loop reverse transcription primer species. In some embodiments, the plurality of target-specific stem-loop reverse transcription primers comprise at least 200 target-specific stem-loop reverse transcription primers. In some embodiments, the plurality of target-specific stem-loop reverse transcription primers comprise at least 300 target-specific stem-loop reverse transcription primers. The number of T residues in the oligo-dT-promoter-containing reverse transcription primer can vary, and in some embodiments includes at least 5, at least 10, at least 15, at least 20, and greater than 20 T residues.
In some embodiments, the present teachings provide a device comprising a solid support comprising a plurality of immobilized probes, wherein the plurality of immobilized probes comprises; A) a plurality of probes complementary to, or complementary to the complement of, the plurality of messenger RNAs; and, B) a plurality of probes complementary to, or complementary to the complement of, the plurality of short target nucleic acids and the corresponding zip-code introduced in a reverse transcription reaction. In some embodiments, the immobilized probes comprise PNA. In some embodiments, the immobilized probes comprise DNA. In some embodiments, various analogs can be employed, as discussed supra. In some embodiments, the plurality of probes complementary to, or complementary to the complement of, the plurality of messenger RNAs comprise at least 1000 different probes. In some embodiments, the plurality of probes complementary to, or complementary to the complement of, the plurality of messenger RNAs comprise probes for an entire transcriptome. In some embodiments, the plurality of probes complementary to, or complementary to the complement of, the plurality of short target nucleic acids and the corresponding zip-code introduced in the reverse transcription reaction comprise at least 100 different probes.
In certain embodiments, the present teachings also provide kits designed to expedite performing certain methods. In some embodiments, kits serve to expedite the performance of the methods of interest by assembling two or more components used in carrying out the methods. In some embodiments, kits may contain components in pre-measured unit amounts to minimize the need for measurements by end-users. In some embodiments, kits may include instructions for performing one or more methods of the present teachings. In certain embodiments, the kit components are optimized to operate in conjunction with one another.
Thus, in some embodiments the present teachings provide a kit for quantitating a plurality of short target nucleic acids, and a plurality of messenger RNAs on a single solid support, wherein each short target nucleic acid is 18-25 nucleotides in length, said method comprising; A) a plurality of target-specific stem-loop reverse transcription primers, wherein each of the plurality of target-specific stem-loop reverse transcription primers comprises a 3′ target-specific portion, a zip-code stem, and a promoter; and, B) an oligo-dT-promoter-containing reverse transcription primer. In some embodiments, the kit further comprises a reverse transcriptase. In some embodiments, the kit further comprises dNTPs.
In some embodiments, the kit further comprises DIG-dUTP. In some embodiments, the kit further comprises a solid support, wherein the solid support comprises a plurality of immobilized probes, wherein the plurality of immobilized probes comprise; A) a plurality of probes complementary to, or complementary to the complement of, the plurality of messenger RNAs; and, B) a plurality of probes complementary to, or complementary to the complement of, the plurality of short target nucleic acids and the corresponding zip-code introduced in a reverse transcription reaction. In some embodiments, the kit further comprises immobilized probes that comprise PNA. In some embodiments, the kit further comprises immobilized probes that comprise DNA.
While the present teachings have been described in terms of these exemplary embodiments, the skilled artisan will readily understand that numerous variations and modifications of these exemplary embodiments are possible without undue experimentation. All such variations and modifications are within the scope of the current teachings. Aspects of the present teachings may be further understood in light of the following example, which should not be construed as limiting the scope of the teachings in any way.
Although the disclosed teachings have been described with reference to various applications, methods, kits, and compositions, it will be appreciated that various changes and modifications may be made without departing from the teachings herein and the claimed invention below. The foregoing examples are provided to better illustrate the disclosed teachings and are not intended to limit the scope of the teachings presented herein.
This application is a continuation of U.S. application Ser. No. 11/562,359, filed Nov. 21, 2006, which claims a priority benefit under 35 U.S.C. §119(e) from U.S. Patent Application No. 60/781,208, filed Mar. 10, 2006, U.S. Patent Application No. 60/790,472, filed Apr. 7, 2006 and U.S. Patent Application No. 60/800,376, filed May 15, 2006, which are incorporated herein by reference.
| Number | Date | Country | |
|---|---|---|---|
| 60781208 | Mar 2006 | US | |
| 60790472 | Apr 2006 | US | |
| 60800376 | May 2006 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 11562359 | Nov 2006 | US |
| Child | 12706627 | US |
