WO2000075356A1 - Amplification en chaine d'arn par polymerase - Google Patents

Amplification en chaine d'arn par polymerase Download PDF

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Publication number
WO2000075356A1
WO2000075356A1 PCT/US1999/012461 US9912461W WO0075356A1 WO 2000075356 A1 WO2000075356 A1 WO 2000075356A1 US 9912461 W US9912461 W US 9912461W WO 0075356 A1 WO0075356 A1 WO 0075356A1
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WIPO (PCT)
Prior art keywords
complementary dnas
polynucleotide
tailed
promoter
activity
Prior art date
Application number
PCT/US1999/012461
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English (en)
Inventor
Shi-Lung Lin
Shao-Yao Ying
Cheng-Ming Chuong
Randall Bruce Widelitz
Original Assignee
Lin Shi Lung
Ying Shao Yao
Chuong Cheng Ming
Randall Bruce Widelitz
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Lin Shi Lung, Ying Shao Yao, Chuong Cheng Ming, Randall Bruce Widelitz filed Critical Lin Shi Lung
Priority to AU43323/99A priority Critical patent/AU4332399A/en
Priority to PCT/US1999/012461 priority patent/WO2000075356A1/fr
Publication of WO2000075356A1 publication Critical patent/WO2000075356A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • C12P19/30—Nucleotides
    • C12P19/34—Polynucleotides, e.g. nucleic acids, oligoribonucleotides
    • 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

  • the present invention generally relates to the field of methods for generating amplified messenger RNA sequences. More particularly, the present invention relates to the field of polymerase chain reaction methods of messenger RNA amplification from single cells.
  • Crino et.al. "Embryonic neuronal markers in tuberous sclerosis: Single- Cell Molecular Pathology", Proc. Natl. Acad. Sci. USA 93: 14152-14157 (1996).
  • mRNA messenger RNAs
  • cDNA complementary DNAs
  • RNA-related cDNAs by reverse transcription-polymerase chain reaction (RT-PCR) has become the most common way among current RNA amplification methods.
  • RT-PCR reverse transcription-polymerase chain reaction
  • Prior art attempts at amplifying RNAs with RT-PCR such as United States Patent No. 4,683,202 to Mullis and United States Patent No. 5,817,465 to Mallet, uses mRNA templates to multiply respective cDNAs.
  • the RT-PCR methods successfully increase the quantity of double-stranded cDNAs from respective RNAs, the fidelity of resulting cDNAs is not completely identical to their RNA origins due to the mis-reading nature of polymerase chain reaction (PCR).
  • RT-PCR the final products of RT-PCR are double-stranded DNAs, rather than mRNAs, which can not be used in probe hybridization and in vitro translation.
  • current RT-PCR methods are not designed for the amplification of full-length mRNAs or cDNAs, indicating the impossibility of generating a full-length cDNA library by this method.
  • Those disadvantages exclude the use of RT-PCR in the analysis of screening unknown gene expressions.
  • aRNA amplified anti-sense RNAs
  • the generation of amplified anti-sense RNAs has been developed to increase transcriptional copies of specific mRNAs from single cells (Van Gelder et.al., Proc. Natl. Acad. Sci. USA 87: 1663-1667 (1990)).
  • the aRNA can be used for characterization of the cellular expression pattern of certain genes, but not all (O'Dell et.al., BioTechniques 25: 566-570 (1998)).
  • Prior art attempts at aRNA amplification such as United States Patent No. 5,514,545 to Eberwine, uses the amplified aRNAs as probes for potential diagnosis and therapy.
  • oligo(dT)-promoter By incorporating a poly(dT) primer coupled to a T7 RNA polymerase promoter sequence, named oligo(dT)-promoter, during reverse transcription (RT), the single copy mRNA can be amplified up to two thousand folds by one round of aRNA amplification (Eberwine et.al. , Proc. Natl. Acad. Sci. USA 89: 3010-3014 (1992)).
  • the aRNAs prepared from single live neuron has been reported to cover 50-75% of total intracellular mRNA population (Eberwine et.al., (1992); Crino et.al., Proc. Natl. Acad. Sci.
  • the present invention is a novel polymerase chain reaction method which amplifies messenger RNAs from single cells.
  • a preferred embodiment of the present invention method includes the following steps:
  • the cycling steps of (d) through (f) can be repeated at least one time for the amplification of said messenger RNAs.
  • the final nucleotide products are preserved in the form of double-stranded duplexes to prevent the degradation of amplified messenger RNAs, preferably, in the form of RNA-DNA hybrid duplexes in the step (f).
  • the mRNAs can be prepared from a plurality of fixed cells, wherein said fixed cells are protected from RNA degradation and also subjected to permeabilisation for enzyme penetration.
  • Those fixed cells include fixative-treated cultural cells, frozen fresh tissues, fixative-treated fresh tissues or paraffin-embedded tissues on slides.
  • the promoter sequences are preferably incorporated into the 5 '-ends of said second-strand cDNAs.
  • said amplified mRNAs are preferably capped by P - 5 ' -(7-methyl)-guanosine-P 3 -5 ' -adenosine-triphosphate or P ' -5 '-(7-methyl)-guanosine- P 3 -5'-guanosine-triphosphate in the step (e) for further in vitro translation.
  • the deoxynucleotide used in the tailing reaction of said first-strand complementary DNAs is either deoxyguanylate (dG) or deoxycytidylate (dC), and the average number of tailed nucleotides is larger than seven; most preferably, the number is about twelve.
  • the final amplified mRNAs can be continuously reverse-transcribed into double-stranded cDNA by Tth-like DNA polymerase activity.
  • the final double-stranded cDNAs are preferably cloned into competent vectors for further applications, such as transfection assay, differential screening, functional detection and so on.
  • FIG.l is an illustration of the preferred embodiment of RNA-polymerase chain reaction of the subject invention.
  • FIG.2 is an illustration of second preferred embodiment of RNA-polymerase chain reaction of the subject invention
  • FIG.3 is an illustration of third preferred embodiment of the RNA-polymerase chain reaction of the subject invention.
  • FIGS.4a-4b is the result of example 4 of the subject invention.
  • RNA-polymerase chain reaction RNA-polymerase chain reaction
  • This method is primarily designed for differential screening of tissue- specific gene expressions in cell level, cloning full-length sequences of unknown gene transcripts, generating pure probes for hybridization assays, synthesizing peptides in vitro, and preparing complete cDNA libraries for gene chip technology.
  • the purpose of the RNA-PCR relies on the repeating steps of reverse transcription, denaturation, double-stranded cDNA synthesis and in vitro transcription to bring up the population of mRNAs to two thousand folds in one cycle of above procedure.
  • the preferred version (FIG.l) of the present invention is based on: 1) prevention of mRNA degradation, 2) first reverse transcription and terminal transferase reaction to incorporate 3 '-polynucleotide tails to the first-strand cDNAs, 3) denaturation and then double-stranded cDNA formation based on the extension of specific promoter-primers complementary to the 3 '-polynucleotide tails, 4) transcription from promoter to amplify mRNAs up to two thousand folds per round, and 5) repeating aforementioned steps to achieve desired RNA amplification.
  • the second preferred version (FIG.2) of the present invention is based on: 1) prevention of mRNA degradation, 2) first reverse transcription to incorporate first promoters to the 5 '-ends of first-strand cDNAs and then addition of polynucleotide sequences to the 3 '-ends of the first-strand cDNAs, 3) double-stranded cDNA synthesis based on the extension of second promoter sequences complementary to the 3 '-polynucleotide regions of the first-strand cDNAs, 4) transcription to amplify either aRNAs or mRNAs up to two thousand folds in the first round of amplification cycle, and 5) repeating aforementioned cycling steps to achieve desired amount of RNAs.
  • the first promoter used here is different from the second promoter, resulting the control of transcription by adding different RNA polymerases.
  • the first promoter is incorporated for aRNA amplification, whereas the second promoter is designed for mRNA amplification.
  • the second and third preferred embodiments are more complicated than the first preferred embodiment (FIG.l), the principle and broad features of the second and third preferred embodiments are completely within the scope of the first preferred embodiment of the present invention.
  • the first-strand complementary DNA refers to a DNA sequence which is complementary to a messenger RNA sequence in an A-T and C-G composition.
  • the anti-sense RNA refers to a RNA sequence which is complementary to a messenger RNA sequence in an A-U and C-G composition.
  • the oligo(dT)-promoter sequence refers to an RNA polymerase promoter sequence coupled with a poly-deoxythymidylate (dT) sequence in its 3 '-end, of which the minimal number of linked dT is seven; most preferably, the number is about twenty-six.
  • the sense sequence refers to a nucleotide sequence which is in the same sequence and composition as its homologue in mRNAs
  • the anti-sense sequence refers to a nucleotide sequence which is complementary to its respective mRNA homologue.
  • the oligo(anti-sense polynucleotide)-promoter sequence refers to an oligonucleotide sequence which is complementary to the polynucleotide-tail of said polynucleotide-tailed cDNA and also linked to an RNA polymerase promoter in its 5'- end.
  • the cycle preferably starts from reverse transcription of mRNAs with Tth-like DNA polymerase, following a tailing reaction with terminal transferase and then denaturation of resulting RNA-cDNA hybrid duplexes. After renaturation of above cDNAs to a specific primer or promoter primer, double-stranded cDNAs are formed by Tth-like DNA polymerase. And then, promoter- specific RNA polymerase is added to accomplish the transcriptional amplification of mRNAs.
  • the advantages of this amplification cycle of the present invention are as follows: 1) single copy mRNAs can be increased to 2000 folds in one round of amplification without mis-reading mistakes, 2) the mRNA amplification is linear and does not result in preferential amplification of abundant mRNA species, 3) the mRNA degradation is inhibited by using fixed cells, and 4) the final mRNA products can be full-length and directly used to generate a complete cDNA library.
  • the amplification of aRNAs and mRNAs can be separated by adding different RNA polymerases in the step (e), but not both.
  • the third preferred embodiment referring to FIG.
  • thermostable RNA polymerases become available, the amplification cycle can be directly completed in a microtube by following the first preferred embodiment (FIG.l) of the present invention.
  • FIG.l first preferred embodiment
  • sequence-specific primers and sequence-specific promoter-linked primers to accomplish the amplification of normalized aRNAs, mRNAs, first-strand cDNAs and second-strand cDNAs of interest.
  • the labeling of cDNAs is accomplished by incorporation of labeled nucleotides or analogs during reverse transcription of Tth-like DNA polymerase activity, while that of the RNAs is completed during transcription .
  • the nucleotide sequences so generated are capable of being probes in a variety of applications, such as Northern blots, Southern blots, dot hybridization, in situ hybridization, position cloning, anti-sense knock-out transfection and so on.
  • the preferred embodiments (FIGS.l, 2 and 3) provide amplified full-length mRNAs for in vitro translation.
  • a cap- nucleotide can be added to the 5 '-end of amplified mRNAs during the transcription step of the present invention.
  • the capped mRNAs can be directly used in protein synthesis and may help the isolation of such protein.
  • the preferred cap- nucleotides include P 1 -5'-(7-methyl)-guanosine-P 3 -5'-adenosine-triphosphate and P 1 - 5'-(7-methyl)-guanosine-P 3 -5'-guanosine-triphosphate.
  • the first step of the present invention can start from fixed cells as well as mRNAs; i.e., fixed cultured cells, frozen fresh tissues, fixed tissues or tissues in slides. Since this formed mRNAs are of full-length and carry RNA promoter regions for in vitro/vivo expression, the transfection of certain gene transcript can be directly performed after its respective double-stranded cDNA is cloned into a competent vector. On the other hand, the present invention are also very useful in preparing complete full-length cDNA libraries for modern gene chip technology.
  • tissue-specific cDNA libraries based on special cell types can be formed and transferred onto a filter, membrane or chip for preserving these genetic information.
  • the cDNA-encoded gene chips may function as an individual source for differential screening, pathological diagnosis, physiological prognosis and genetic identification. This kind of approach will become more and more important following the completion of human genome project in the year 2003.
  • RNA-PCR As shown in FIG.l, 2 and 3 of the present invention, according to the high amplification rate of RNA polymerase (about 2000 folds/cycle), the labor- and time-consuming factors in this RNA-PCR can be reduced to the minimum. Also, the preparation of amplified mRNAs is cheaper and more efficient than poly(dT)-linked chromatography columns in previous methods. Most importantly, this RNA amplification can be carried out in microtubes with only few cells. Taken together, these special features make the content of RNA-PCR as simple, fast, and inexpensive as a kit for concisely isolating amplified mRNA sequences of interest.
  • LNCaP cells a prostate cancer cell line
  • RPMI 1640 medium supplemented with 2% fetal calf serum.
  • One 70% full of cells cultured in 60mm dish were trypsinized, collected and washed three times in 5ml phosphate buffered saline (PBS, pH 7.2) at room temperature, then suspended in 1ml of ice-cold 10% formaldehyde solution in 0.15M NaCl. After one hour incubation on ice with occasional agitation, the cells were centrifuged at 13,000rpm for 2 min and wash three times in ice-cold PBS with vigorous pipetting.
  • PBS phosphate buffered saline
  • RNAs in cells For first reverse transcription of mRNAs in cells, one hundred of the fixed cells were thawed, resuspended in 20 ⁇ l of DEPC-treated ddH 2 O, mixed with 25pmol oligo(dT)-T7 promoter (SEQ ID.1), heated to 65°C for 5 min and then cooled on ice. A 50 ⁇ l RT reaction was prepared, comprising lO ⁇ l of 5x Mg-containing RT buffer (Boehringer Mannheim), 2mM dNTPs, RNase inhibitor and above cooled cells. After C.
  • therm, polymerase (5U) was added, the RT reaction was mixed and incubated at 55°C for 10 min and shifted to 72°C for one hour, and then the cells were washed once with PBS and resuspended in a 50 ⁇ l tailing reaction, comprising 2mM dGTP, lO ⁇ l of 5x tailing buffer (250mM KC1, 50mM Tris-HCl, 8mM MgCl 2 , pH 8.3 at 20°C). The tailing reaction was heated at 94°C for 3 min and then chilled in ice for mixing with terminal transferase (20U), following further incubation at 37°C for 20 min. This formed said polynucleotide-tailed first-strand cDNAs.
  • a transcription reaction (50 ⁇ l) was prepared, containing 5 ⁇ l of lOx transcription buffer (Boehringer Mannheim), 2mM rNTPs, RNA inhibitor and T7 RNA polymerase (2000U). After three hour incubation at 37°C, the cDNA transcripts were isolated from both cells and supernatant, and can be directly used in following reverse transcription. The reaction was finally stopped at 94°C for 3 min and chilled in ice immediately.
  • a 50 ⁇ l RT reaction was prepared, comprising lO ⁇ l of 5x Mg-containing RT buffer (Boehringer Mannheim), 25pmol oligo(dC)-SP6 promoter primer (SEQ ID.3), 2mM dNTPs, RNase inhibitor and 5 ⁇ l of above aRNA-containing supernatant.
  • polymerase 5U
  • the RT reaction was mixed and incubated at 55°C for 10 min and shifted to 72°C for one hour. This formed said second-strand cDNAs.
  • double-stranded cDNAs can be formed by adding ImM dNTPs at 70°C for 5 min.
  • a transcription reaction 50 ⁇ l was then prepared to generate said amplified mRNAs, containing 5 ⁇ l of lOx transcription buffer (Boehringer Mannheim), 2mM rNTPs, RNA inhibitor and SP6 RNA polymerase (2000U). After three hour incubation at 37°C, the cDNA transcripts were isolated and can be directly used in another round of RNA-PCR. The final reaction was stopped at 94°C for 3 min and chilled in ice immediately..
  • the present invention is a polymerase chain reaction method of generating amplified messenger RNAs from single cells, comprising the steps of:
  • the present invention is an RNA amplification method of performing improved messenger RNA enrichment, comprising the steps of:

Abstract

La présente invention concerne une méthode rapide, simple et spécifique de production d'ARN messagers amplifiés à partir d'ARN messagers limités. Le principe de cette méthode d'amplification en chaîne d'ARN par polymérase se base sur les étapes cycliques de la transcription inverse, dénaturation, synthèse d'ADNc bicaténaire et ensuite la transcription in vitro afin d'élever la quantité d'ARN messagers de 2000 fois en un cycle de la procédure précitée. Cette méthode est conçue essentiellement pour le criblage différentiel d'expressions de gènes à spécificité tissulaire au niveau cellulaire, le clonage de séquence de longueur totale de transcrits de gène inconnu, la production de sondes destinées à des dosages d'hybridation, la synthèse de peptides in vitro et la préparation d'ADNc représentatif pour la technologie moderne des puces à ADN. Simultanément à une étape de fixation et de perméabilisation cellulaire, une banque d'ADNc de longueur totale complète peut être générée directement à partir de quelques cellules individuelles sans dégradation de l'ARNm.
PCT/US1999/012461 1999-06-04 1999-06-04 Amplification en chaine d'arn par polymerase WO2000075356A1 (fr)

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AU43323/99A AU4332399A (en) 1999-06-04 1999-06-04 Rna polymerase chain reaction
PCT/US1999/012461 WO2000075356A1 (fr) 1999-06-04 1999-06-04 Amplification en chaine d'arn par polymerase

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Cited By (36)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002064835A2 (fr) * 2001-01-31 2002-08-22 Ambion, Inc. Methodes d'analyse d'empreintes d'acides nucleiques
EP1241268A2 (fr) * 2001-03-12 2002-09-18 Nisshinbo Industries, Inc. Méthode d'amplification d'ARN
EP1275738A1 (fr) * 2001-07-11 2003-01-15 Roche Diagnostics GmbH Procédé pour la synthèse aléatoire et l'amplification d'ADNc
WO2003020873A2 (fr) * 2001-09-03 2003-03-13 Artus - Gesellschaft Für Molekularbiologische Diagnostik Und Entwicklung Mbh Multiplication d'acides ribonucleiques
EP1371726A1 (fr) * 2002-06-14 2003-12-17 Rijksuniversiteit Groningen Procédé d'amplification d'ARN
EP1392866A2 (fr) * 2001-05-14 2004-03-03 Henry Hongjun Ji Nouveau procede pour cloner les sequences a domaine variable du repertoire des genes immunologiques
WO2004048596A2 (fr) 2002-11-21 2004-06-10 Epicentre Technologies Procedes d'utilisation d'amorces qui codent un brin d'un promoteur bicatenaire
EP1485501A2 (fr) * 2002-02-22 2004-12-15 Ortho-McNeil Pharmaceutical, Inc. Procede pour generer l'arn amplifie
WO2007120863A2 (fr) 2006-04-14 2007-10-25 Epicentre Technologies Kits et procedes pour la generation d'arn coiffe en 5'
WO2008141561A1 (fr) * 2007-05-17 2008-11-27 Wondergen Bio-Medicine Technology Co., Ltd. Pcr de substitut de système
WO2009006438A2 (fr) 2007-06-29 2009-01-08 Epicentre Technologies Corporation Adn de copie et arn sens
US7838270B2 (en) 2001-05-22 2010-11-23 The University Of Chicago Target-dependent transcription using deletion mutants of N4 RNA polymerase
US8137911B2 (en) 2001-05-22 2012-03-20 Cellscript, Inc. Preparation and use of single-stranded transcription substrates for synthesis of transcription products corresponding to target sequences
US8946177B2 (en) 2004-11-12 2015-02-03 Mima Therapeutics, Inc Methods and compositions involving miRNA and miRNA inhibitor molecules
US8980864B2 (en) 2013-03-15 2015-03-17 Moderna Therapeutics, Inc. Compositions and methods of altering cholesterol levels
US8999380B2 (en) 2012-04-02 2015-04-07 Moderna Therapeutics, Inc. Modified polynucleotides for the production of biologics and proteins associated with human disease
US9057100B2 (en) 2001-06-30 2015-06-16 Enzo Life Sciences, Inc. Composition comprising array of nucleic acid primer sets
US9068948B2 (en) 2002-03-12 2015-06-30 Enzo Life Sciences, Inc. Processes for detection of nucleic acids
US9080215B2 (en) 2007-09-14 2015-07-14 Asuragen, Inc. MicroRNAs differentially expressed in cervical cancer and uses thereof
US9107886B2 (en) 2012-04-02 2015-08-18 Moderna Therapeutics, Inc. Modified polynucleotides encoding basic helix-loop-helix family member E41
US9181319B2 (en) 2010-08-06 2015-11-10 Moderna Therapeutics, Inc. Engineered nucleic acids and methods of use thereof
US9186372B2 (en) 2011-12-16 2015-11-17 Moderna Therapeutics, Inc. Split dose administration
US9283287B2 (en) 2012-04-02 2016-03-15 Moderna Therapeutics, Inc. Modified polynucleotides for the production of nuclear proteins
US9334328B2 (en) 2010-10-01 2016-05-10 Moderna Therapeutics, Inc. Modified nucleosides, nucleotides, and nucleic acids, and uses thereof
US9365852B2 (en) 2008-05-08 2016-06-14 Mirna Therapeutics, Inc. Compositions and methods related to miRNA modulation of neovascularization or angiogenesis
US9428535B2 (en) 2011-10-03 2016-08-30 Moderna Therapeutics, Inc. Modified nucleosides, nucleotides, and nucleic acids, and uses thereof
US9464124B2 (en) 2011-09-12 2016-10-11 Moderna Therapeutics, Inc. Engineered nucleic acids and methods of use thereof
US9533047B2 (en) 2011-03-31 2017-01-03 Modernatx, Inc. Delivery and formulation of engineered nucleic acids
US9572897B2 (en) 2012-04-02 2017-02-21 Modernatx, Inc. Modified polynucleotides for the production of cytoplasmic and cytoskeletal proteins
US9597380B2 (en) 2012-11-26 2017-03-21 Modernatx, Inc. Terminally modified RNA
US9644241B2 (en) 2011-09-13 2017-05-09 Interpace Diagnostics, Llc Methods and compositions involving miR-135B for distinguishing pancreatic cancer from benign pancreatic disease
US9777312B2 (en) 2001-06-30 2017-10-03 Enzo Life Sciences, Inc. Dual polarity analysis of nucleic acids
US10047388B2 (en) 2004-05-28 2018-08-14 Asuragen, Inc. Methods and compositions involving MicroRNA
US10323076B2 (en) 2013-10-03 2019-06-18 Modernatx, Inc. Polynucleotides encoding low density lipoprotein receptor
US10815291B2 (en) 2013-09-30 2020-10-27 Modernatx, Inc. Polynucleotides encoding immune modulating polypeptides
US11926817B2 (en) 2019-08-09 2024-03-12 Nutcracker Therapeutics, Inc. Microfluidic apparatus and methods of use thereof

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5501963A (en) * 1992-09-11 1996-03-26 Hoffmann-La Roche Inc. Amplification and detection of nucleic acids in blood samples
US5599672A (en) * 1992-03-11 1997-02-04 Dana-Farber Cancer Institute, Inc. Method of differential display of exposed mRNA by RT/PCR

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5599672A (en) * 1992-03-11 1997-02-04 Dana-Farber Cancer Institute, Inc. Method of differential display of exposed mRNA by RT/PCR
US5665547A (en) * 1992-03-11 1997-09-09 Dana Farber Cancer Institute Methods of comparing levels or amounts of mRNAs
US5501963A (en) * 1992-09-11 1996-03-26 Hoffmann-La Roche Inc. Amplification and detection of nucleic acids in blood samples

Cited By (100)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002064835A2 (fr) * 2001-01-31 2002-08-22 Ambion, Inc. Methodes d'analyse d'empreintes d'acides nucleiques
WO2002064835A3 (fr) * 2001-01-31 2003-12-11 Ambion Inc Methodes d'analyse d'empreintes d'acides nucleiques
EP1241268A2 (fr) * 2001-03-12 2002-09-18 Nisshinbo Industries, Inc. Méthode d'amplification d'ARN
EP1241268A3 (fr) * 2001-03-12 2003-10-15 Nisshinbo Industries, Inc. Méthode d'amplification d'ARN
EP1392866A2 (fr) * 2001-05-14 2004-03-03 Henry Hongjun Ji Nouveau procede pour cloner les sequences a domaine variable du repertoire des genes immunologiques
EP1392866A4 (fr) * 2001-05-14 2004-12-22 Henry Hongjun Ji Nouveau procede pour cloner les sequences a domaine variable du repertoire des genes immunologiques
US8137911B2 (en) 2001-05-22 2012-03-20 Cellscript, Inc. Preparation and use of single-stranded transcription substrates for synthesis of transcription products corresponding to target sequences
US7838270B2 (en) 2001-05-22 2010-11-23 The University Of Chicago Target-dependent transcription using deletion mutants of N4 RNA polymerase
US9777312B2 (en) 2001-06-30 2017-10-03 Enzo Life Sciences, Inc. Dual polarity analysis of nucleic acids
US9057100B2 (en) 2001-06-30 2015-06-16 Enzo Life Sciences, Inc. Composition comprising array of nucleic acid primer sets
US9279147B2 (en) 2001-06-30 2016-03-08 Enzo Life Sciences, Inc. Processes for detecting or quantifying analytes of interest
US9650666B2 (en) 2001-06-30 2017-05-16 Enzo Biochem, Inc. Processes for detecting or quantifying nucleic acids using an array of fixed or immobilized nucleic acids
US9487821B2 (en) 2001-06-30 2016-11-08 Enzo Life Sciences, Inc. Composition comprising library of double stranded nucleic acids
US9528146B2 (en) 2001-06-30 2016-12-27 Enzo Life Sciences, Inc. Processes for detecting or quantifying more than one nucleic acid in a library
US9617584B2 (en) 2001-06-30 2017-04-11 Enzo Biochem, Inc. Processes for detecting or quantifying nucleic acids using an array of fixed or immobilized nucleic acids
US9434984B2 (en) 2001-06-30 2016-09-06 Enzo Life Sciences, Inc. Composition comprising an array which further comprises chimeric compositions
US9428797B2 (en) 2001-06-30 2016-08-30 Enzo Life Sciences, Inc. Nucleic acid detecting or quantifying processes
US9309563B2 (en) 2001-06-30 2016-04-12 Enzo Life Sciences, Inc. Compositions and processes for analyte detection, quantification and amplification
US9234234B2 (en) 2001-06-30 2016-01-12 Enzo Life Sciences, Inc. Detection and quantification process for more than one nucleic acid in library
US9873956B2 (en) 2001-06-30 2018-01-23 Enzo Biochem, Inc. Compositions and processes for analyte detection, quantification and amplification
US9163280B2 (en) 2001-06-30 2015-10-20 Enzo Life Sciences, Inc. Process for detecting or quantifying nucleic acids in a library
US9234235B2 (en) 2001-06-30 2016-01-12 Enzo Life Sciences, Inc. Processes for detecting or quantifying nucleic acids using an array of fixed or immobilized nucleic acids
EP1275738A1 (fr) * 2001-07-11 2003-01-15 Roche Diagnostics GmbH Procédé pour la synthèse aléatoire et l'amplification d'ADNc
WO2003020873A2 (fr) * 2001-09-03 2003-03-13 Artus - Gesellschaft Für Molekularbiologische Diagnostik Und Entwicklung Mbh Multiplication d'acides ribonucleiques
WO2003020873A3 (fr) * 2001-09-03 2003-10-23 Artus Ges Fuer Molekularbiolog Multiplication d'acides ribonucleiques
US7354742B2 (en) 2002-02-22 2008-04-08 Ortho-Mcneil Pharmaceutical, Inc. Method for generating amplified RNA
EP1485501A4 (fr) * 2002-02-22 2005-12-28 Ortho Mcneil Pharm Inc Procede pour generer l'arn amplifie
EP1485501A2 (fr) * 2002-02-22 2004-12-15 Ortho-McNeil Pharmaceutical, Inc. Procede pour generer l'arn amplifie
US9261460B2 (en) 2002-03-12 2016-02-16 Enzo Life Sciences, Inc. Real-time nucleic acid detection processes and compositions
US9068948B2 (en) 2002-03-12 2015-06-30 Enzo Life Sciences, Inc. Processes for detection of nucleic acids
US9316587B2 (en) 2002-03-12 2016-04-19 Enzo Life Sciences, Inc. Processes for quantitative or qualitative detection of single-stranded or double-stranded nucleic acids
EP1371726A1 (fr) * 2002-06-14 2003-12-17 Rijksuniversiteit Groningen Procédé d'amplification d'ARN
WO2004048596A2 (fr) 2002-11-21 2004-06-10 Epicentre Technologies Procedes d'utilisation d'amorces qui codent un brin d'un promoteur bicatenaire
US10047388B2 (en) 2004-05-28 2018-08-14 Asuragen, Inc. Methods and compositions involving MicroRNA
US9447414B2 (en) 2004-11-12 2016-09-20 Asuragen, Inc. Methods and compositions involving miRNA and miRNA inhibitor molecules
US9506061B2 (en) 2004-11-12 2016-11-29 Asuragen, Inc. Methods and compositions involving miRNA and miRNA inhibitor molecules
US8946177B2 (en) 2004-11-12 2015-02-03 Mima Therapeutics, Inc Methods and compositions involving miRNA and miRNA inhibitor molecules
US9382537B2 (en) 2004-11-12 2016-07-05 Asuragen, Inc. Methods and compositions involving miRNA and miRNA inhibitor molecules
US9051571B2 (en) 2004-11-12 2015-06-09 Asuragen, Inc. Methods and compositions involving miRNA and miRNA inhibitor molecules
US9068219B2 (en) 2004-11-12 2015-06-30 Asuragen, Inc. Methods and compositions involving miRNA and miRNA inhibitor molecules
US9115380B2 (en) 2006-04-14 2015-08-25 Cellscript, Llc Kits and methods for generating 5′ capped RNA
US8846348B2 (en) 2006-04-14 2014-09-30 Cellscript, Llc Kits and methods for generating 5′ capped RNA
US9005930B2 (en) 2006-04-14 2015-04-14 Cellscript, Llc Kits and methods for generating 5′ capped RNA
WO2007120863A2 (fr) 2006-04-14 2007-10-25 Epicentre Technologies Kits et procedes pour la generation d'arn coiffe en 5'
WO2008141561A1 (fr) * 2007-05-17 2008-11-27 Wondergen Bio-Medicine Technology Co., Ltd. Pcr de substitut de système
WO2009006438A2 (fr) 2007-06-29 2009-01-08 Epicentre Technologies Corporation Adn de copie et arn sens
US8039214B2 (en) 2007-06-29 2011-10-18 Cellscript, Inc. Synthesis of tagged nucleic acids
US8329887B2 (en) 2007-06-29 2012-12-11 Cellscript, Inc. Synthesis of tagged nucleic acids
US9080215B2 (en) 2007-09-14 2015-07-14 Asuragen, Inc. MicroRNAs differentially expressed in cervical cancer and uses thereof
US9365852B2 (en) 2008-05-08 2016-06-14 Mirna Therapeutics, Inc. Compositions and methods related to miRNA modulation of neovascularization or angiogenesis
US9937233B2 (en) 2010-08-06 2018-04-10 Modernatx, Inc. Engineered nucleic acids and methods of use thereof
US9181319B2 (en) 2010-08-06 2015-11-10 Moderna Therapeutics, Inc. Engineered nucleic acids and methods of use thereof
US9447164B2 (en) 2010-08-06 2016-09-20 Moderna Therapeutics, Inc. Engineered nucleic acids and methods of use thereof
US9657295B2 (en) 2010-10-01 2017-05-23 Modernatx, Inc. Modified nucleosides, nucleotides, and nucleic acids, and uses thereof
US9334328B2 (en) 2010-10-01 2016-05-10 Moderna Therapeutics, Inc. Modified nucleosides, nucleotides, and nucleic acids, and uses thereof
US10064959B2 (en) 2010-10-01 2018-09-04 Modernatx, Inc. Modified nucleosides, nucleotides, and nucleic acids, and uses thereof
US9533047B2 (en) 2011-03-31 2017-01-03 Modernatx, Inc. Delivery and formulation of engineered nucleic acids
US9950068B2 (en) 2011-03-31 2018-04-24 Modernatx, Inc. Delivery and formulation of engineered nucleic acids
US9464124B2 (en) 2011-09-12 2016-10-11 Moderna Therapeutics, Inc. Engineered nucleic acids and methods of use thereof
US10751386B2 (en) 2011-09-12 2020-08-25 Modernatx, Inc. Engineered nucleic acids and methods of use thereof
US10022425B2 (en) 2011-09-12 2018-07-17 Modernatx, Inc. Engineered nucleic acids and methods of use thereof
US10655184B2 (en) 2011-09-13 2020-05-19 Interpace Diagnostics, Llc Methods and compositions involving miR-135b for distinguishing pancreatic cancer from benign pancreatic disease
US9644241B2 (en) 2011-09-13 2017-05-09 Interpace Diagnostics, Llc Methods and compositions involving miR-135B for distinguishing pancreatic cancer from benign pancreatic disease
US9428535B2 (en) 2011-10-03 2016-08-30 Moderna Therapeutics, Inc. Modified nucleosides, nucleotides, and nucleic acids, and uses thereof
US9271996B2 (en) 2011-12-16 2016-03-01 Moderna Therapeutics, Inc. Formulation and delivery of PLGA microspheres
US9186372B2 (en) 2011-12-16 2015-11-17 Moderna Therapeutics, Inc. Split dose administration
US9295689B2 (en) 2011-12-16 2016-03-29 Moderna Therapeutics, Inc. Formulation and delivery of PLGA microspheres
US9254311B2 (en) 2012-04-02 2016-02-09 Moderna Therapeutics, Inc. Modified polynucleotides for the production of proteins
US9061059B2 (en) 2012-04-02 2015-06-23 Moderna Therapeutics, Inc. Modified polynucleotides for treating protein deficiency
US9301993B2 (en) 2012-04-02 2016-04-05 Moderna Therapeutics, Inc. Modified polynucleotides encoding apoptosis inducing factor 1
US9283287B2 (en) 2012-04-02 2016-03-15 Moderna Therapeutics, Inc. Modified polynucleotides for the production of nuclear proteins
US9255129B2 (en) 2012-04-02 2016-02-09 Moderna Therapeutics, Inc. Modified polynucleotides encoding SIAH E3 ubiquitin protein ligase 1
US9233141B2 (en) 2012-04-02 2016-01-12 Moderna Therapeutics, Inc. Modified polynucleotides for the production of proteins associated with blood and lymphatic disorders
US9220792B2 (en) 2012-04-02 2015-12-29 Moderna Therapeutics, Inc. Modified polynucleotides encoding aquaporin-5
US9572897B2 (en) 2012-04-02 2017-02-21 Modernatx, Inc. Modified polynucleotides for the production of cytoplasmic and cytoskeletal proteins
US9587003B2 (en) 2012-04-02 2017-03-07 Modernatx, Inc. Modified polynucleotides for the production of oncology-related proteins and peptides
US8999380B2 (en) 2012-04-02 2015-04-07 Moderna Therapeutics, Inc. Modified polynucleotides for the production of biologics and proteins associated with human disease
US9220755B2 (en) 2012-04-02 2015-12-29 Moderna Therapeutics, Inc. Modified polynucleotides for the production of proteins associated with blood and lymphatic disorders
US9221891B2 (en) 2012-04-02 2015-12-29 Moderna Therapeutics, Inc. In vivo production of proteins
US9216205B2 (en) 2012-04-02 2015-12-22 Moderna Therapeutics, Inc. Modified polynucleotides encoding granulysin
US9192651B2 (en) 2012-04-02 2015-11-24 Moderna Therapeutics, Inc. Modified polynucleotides for the production of secreted proteins
US9675668B2 (en) 2012-04-02 2017-06-13 Moderna Therapeutics, Inc. Modified polynucleotides encoding hepatitis A virus cellular receptor 2
US9149506B2 (en) 2012-04-02 2015-10-06 Moderna Therapeutics, Inc. Modified polynucleotides encoding septin-4
US9782462B2 (en) 2012-04-02 2017-10-10 Modernatx, Inc. Modified polynucleotides for the production of proteins associated with human disease
US9814760B2 (en) 2012-04-02 2017-11-14 Modernatx, Inc. Modified polynucleotides for the production of biologics and proteins associated with human disease
US9828416B2 (en) 2012-04-02 2017-11-28 Modernatx, Inc. Modified polynucleotides for the production of secreted proteins
US9827332B2 (en) 2012-04-02 2017-11-28 Modernatx, Inc. Modified polynucleotides for the production of proteins
US9114113B2 (en) 2012-04-02 2015-08-25 Moderna Therapeutics, Inc. Modified polynucleotides encoding citeD4
US9878056B2 (en) 2012-04-02 2018-01-30 Modernatx, Inc. Modified polynucleotides for the production of cosmetic proteins and peptides
US9107886B2 (en) 2012-04-02 2015-08-18 Moderna Therapeutics, Inc. Modified polynucleotides encoding basic helix-loop-helix family member E41
US9095552B2 (en) 2012-04-02 2015-08-04 Moderna Therapeutics, Inc. Modified polynucleotides encoding copper metabolism (MURR1) domain containing 1
US9089604B2 (en) 2012-04-02 2015-07-28 Moderna Therapeutics, Inc. Modified polynucleotides for treating galactosylceramidase protein deficiency
US9303079B2 (en) 2012-04-02 2016-04-05 Moderna Therapeutics, Inc. Modified polynucleotides for the production of cytoplasmic and cytoskeletal proteins
US9050297B2 (en) 2012-04-02 2015-06-09 Moderna Therapeutics, Inc. Modified polynucleotides encoding aryl hydrocarbon receptor nuclear translocator
US10501512B2 (en) 2012-04-02 2019-12-10 Modernatx, Inc. Modified polynucleotides
US9597380B2 (en) 2012-11-26 2017-03-21 Modernatx, Inc. Terminally modified RNA
US8980864B2 (en) 2013-03-15 2015-03-17 Moderna Therapeutics, Inc. Compositions and methods of altering cholesterol levels
US10815291B2 (en) 2013-09-30 2020-10-27 Modernatx, Inc. Polynucleotides encoding immune modulating polypeptides
US10323076B2 (en) 2013-10-03 2019-06-18 Modernatx, Inc. Polynucleotides encoding low density lipoprotein receptor
US11926817B2 (en) 2019-08-09 2024-03-12 Nutcracker Therapeutics, Inc. Microfluidic apparatus and methods of use thereof

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