WO2005058933A1 - Formulations and methods for denaturing proteins - Google Patents
Formulations and methods for denaturing proteins Download PDFInfo
- Publication number
- WO2005058933A1 WO2005058933A1 PCT/US2004/042044 US2004042044W WO2005058933A1 WO 2005058933 A1 WO2005058933 A1 WO 2005058933A1 US 2004042044 W US2004042044 W US 2004042044W WO 2005058933 A1 WO2005058933 A1 WO 2005058933A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- rna
- formulation
- solid support
- alcohol
- concentration
- Prior art date
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
-
- 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
- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
- C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
-
- 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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/99—Enzyme inactivation by chemical treatment
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S435/00—Chemistry: molecular biology and microbiology
- Y10S435/814—Enzyme separation or purification
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
- Y10T436/00—Chemistry: analytical and immunological testing
- Y10T436/25—Chemistry: analytical and immunological testing including sample preparation
- Y10T436/25375—Liberation or purification of sample or separation of material from a sample [e.g., filtering, centrifuging, etc.]
- Y10T436/255—Liberation or purification of sample or separation of material from a sample [e.g., filtering, centrifuging, etc.] including use of a solid sorbent, semipermeable membrane, or liquid extraction
Definitions
- RNA DNA rearrangements associated with common leukemias are detected by isolation and identification of abnormal, hybrid RNAs.
- Common methods for analyzing RNA include northern blotting, ribonuclease protection assays (RPAs), reverse transcriptase-polymerase chain reaction (RT-PCR), cDNA preparation for cloning, in vitro translation and microarray analyses. To obtain valid and consistent results from these analyses, it is important that the RNA be purified from other components common to biological materials such as proteins, carbohydrates, lipids and DNA. Summary of the Invention It should be noted that the indefinite articles "a” and “an” and the definite article “the” are used in the present application, as is common in patent applications, to mean one or more unless the context clearly dictates otherwise.
- the present invention provides a formulation for denaturing proteins that contains a lithium salt at a concentration of about 2.5-4.0 M, an alcohol at a concentration of about 25-40% v/v, and a citrate at a concentration of about 25- 100 mM.
- This formulation lacks EDTA, and lacks a chaotropic substance (e.g., guanidinium salt, urea, ammonium, cesium, rubidium, potassium, or iodide salt).
- the lithium salt is lithium chloride or lithium bromide.
- RNA purification methods fall into two general categories, liquid phase and solid phase purification.
- liquid phase purification the RNA remains in the liquid phase, while impurities are removed by processes such as precipitation and/or centrifugation.
- solid phase purification the RNA is bound to a solid support while impurities such as DNA, proteins, and phospholipids are selectively eluted.
- the present solution does not contain hazardous chaotropic substances such as guanidinium salts, urea, etc.
- the Protein Denaturing Fonnulation of the present invention is unique in that it requires no added strong chaotropic substances such as guanidinium salts, urea, etc. Further, the method does not require heat to denature the protein. Guanidinium salts and urea are strong chaotropic salts that disrupt the structure of water and thus tend to decrease the strength of hydrophobic interactions resulting in a drastic effect on other solute molecules. For example, urea, when dissolved in water, disrupts the secondary, tertiary, and quaternary structures of proteins, and subsequently causes dissociation of proteins from RNA.
- the Protein Denaturing Fonnulation should have the following concentrations: (1) alcohol, about 25-40% v/v (such as about 28-35% ethanol, or about 30% ethanol), (2) lithium salt, about 2.5-4.0 M (such as about 3.2-3.8 M LiCl, or about 3.5 M LiCl), and (3) citrate, about 25- 100 mM (such as about 40-75 mM trisodium citrate, or about 50 mM trisodium citrate).
- the inlet ports, breakaway seals and diffuser may be housed in an optional screw-cap.
- an optional diffuser At the bottom of the solid support is an optional diffuser with a pore size suitable for the dispersion and passage of cellular debris, proteins and lipid molecules.
- the diffusers allow for a uniform traversal of biological material across the cross section of the cartridge, and prevent unequal buildup of biological material anywhere above or below the solid support.
- the outlet of the cartridge comes equipped with a protective cap that fits neatly over the tapered barrel.
- the purified RNA is collected in a collection tube that consists of a conical tube with a snap cap for easy and contamination-free storage. The entire vessel can be scaled in size depending on the size of the samples to be processed and the yields needed for subsequent analysis.
- RNAs include, but are not limited to, ribosomal RNA, messenger RNA, transfer RNA, and viral RNA, or combinations thereof, all of which can be recovered over a wide molecular weight range.
- the reagents, methods and kits of the present invention provide substantially pure and undegraded RNA such that the RNA may be used in downstream processes such as RT-PCR and microanay analyses.
- substantially pure means substantially free of protein (e.g., enzymes), such that the RNA can be used in subsequent analyses known to those with skill in the art such as RT-PCR and microanay analyses.
- Enzyme in buffer solution may be subjected to three freeze/thaw cycles and still retain sufficient DNase activity.
- a sample was homogenized, the cells were lysed, and Wash I Solution (Gentra Systems, Inc.) was added as outlined in the manufacturer's RNA purification protocol. 50 ⁇ l DNase was applied to the column. The column was incubated at room temperature for 15 minutes in order to eliminate the presence of any DNA. Next, 200 ⁇ l of Protein Denaturation Formulation (DNase Wash Solution, Gentra Systems Inc.) was added to the column to denature the DNase I enzyme present in the glass fiber column. The column was centrifuged at 13,000 - 16,000 x g for two minutes in order to denature and wash away the DNase I enzyme.
- DNase Wash Solution Gentra Systems Inc.
- Example 3 Determination of Elimination of Enzyme Activity Nucleic acid contacted with the Protein Denaturation Formulation was tested in a PUC 19 Assay. This assay detennines whether supercoiled (double- stranded) DNA is nicked to form circular PUC, as observed on an agarose gel. The results indicated that the DNA was not nicked, showing that the Protein Denaturation Formulation effectively denatured any DNase I that was present in the original sample.
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- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- Biochemistry (AREA)
- Biotechnology (AREA)
- Wood Science & Technology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- General Health & Medical Sciences (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- General Chemical & Material Sciences (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Crystallography & Structural Chemistry (AREA)
- Analytical Chemistry (AREA)
- Plant Pathology (AREA)
- Medicinal Chemistry (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Peptides Or Proteins (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
- Saccharide Compounds (AREA)
Abstract
Description
Claims
Priority Applications (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2006545361A JP4435787B2 (en) | 2003-12-16 | 2004-12-15 | Formulations and methods for denaturing proteins |
DE602004026912T DE602004026912D1 (en) | 2003-12-16 | 2004-12-15 | FORMULATIONS AND METHOD FOR DENATURING PROTEINS |
AT04814250T ATE466080T1 (en) | 2003-12-16 | 2004-12-15 | FORMULATIONS AND METHODS FOR DENATURING PROTEINS |
CA2549985A CA2549985C (en) | 2003-12-16 | 2004-12-15 | Formulations and methods for denaturing proteins |
EP04814250A EP1694692B1 (en) | 2003-12-16 | 2004-12-15 | Formulations and methods for denaturing proteins |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US53039203P | 2003-12-16 | 2003-12-16 | |
US60/530,392 | 2003-12-16 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2005058933A1 true WO2005058933A1 (en) | 2005-06-30 |
Family
ID=34700134
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US2004/042044 WO2005058933A1 (en) | 2003-12-16 | 2004-12-15 | Formulations and methods for denaturing proteins |
Country Status (7)
Country | Link |
---|---|
US (1) | US7115719B2 (en) |
EP (1) | EP1694692B1 (en) |
JP (1) | JP4435787B2 (en) |
AT (1) | ATE466080T1 (en) |
CA (1) | CA2549985C (en) |
DE (1) | DE602004026912D1 (en) |
WO (1) | WO2005058933A1 (en) |
Cited By (14)
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EP1727900A1 (en) * | 2004-03-26 | 2006-12-06 | Fuji Photo Film Co., Ltd. | Method for selectively separating and purifying rna and method for separating and purifying nucleic acid |
JP2007202514A (en) * | 2006-02-03 | 2007-08-16 | Fujifilm Corp | Method for separating and purifying nucleic acid |
WO2009121208A1 (en) * | 2008-04-02 | 2009-10-08 | 芮宝生医股份有限公司 | Reagents, chaotropic agents, kits and methods for isolating nucleic acids based on magnetic cellulose materials |
US7776616B2 (en) | 1997-09-17 | 2010-08-17 | Qiagen North American Holdings, Inc. | Apparatuses and methods for isolating nucleic acid |
EP1960521B1 (en) * | 2005-12-07 | 2011-09-21 | AJ Innuscreen GmbH | Method and test kit for the separation, purification and recovery of long- and short-chain nucleic acids |
WO2014144767A1 (en) * | 2013-03-15 | 2014-09-18 | Moderna Therapeutics, Inc. | Ion exchange purification of mrna |
US10017826B2 (en) | 2015-05-08 | 2018-07-10 | Curevac Ag | Method for producing RNA |
US10077439B2 (en) | 2013-03-15 | 2018-09-18 | Modernatx, Inc. | Removal of DNA fragments in mRNA production process |
US10138507B2 (en) | 2013-03-15 | 2018-11-27 | Modernatx, Inc. | Manufacturing methods for production of RNA transcripts |
US10155785B2 (en) | 2014-04-25 | 2018-12-18 | Translate Bio, Inc. | Methods for purification of messenger RNA |
US10876104B2 (en) | 2013-03-14 | 2020-12-29 | Translate Bio, Inc. | Methods for purification of messenger RNA |
US10947527B2 (en) | 2004-11-05 | 2021-03-16 | Qiagen North American Holdings, Inc | Compositions and methods for purifying nucleic acids from stabilization reagents |
US11174500B2 (en) | 2018-08-24 | 2021-11-16 | Translate Bio, Inc. | Methods for purification of messenger RNA |
US11667910B2 (en) | 2015-05-29 | 2023-06-06 | CureVac Manufacturing GmbH | Method for producing and purifying RNA, comprising at least one step of tangential flow filtration |
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US20050032105A1 (en) * | 2001-10-12 | 2005-02-10 | Bair Robert Jackson | Compositions and methods for using a solid support to purify DNA |
US7148343B2 (en) * | 2001-10-12 | 2006-12-12 | Gentra Systems, Inc. | Compositions and methods for using a solid support to purify RNA |
US7893228B2 (en) * | 2001-10-12 | 2011-02-22 | Qiagen North American Holdings, Inc. | Compositions and methods for using a solid support to purify RNA |
US20070092403A1 (en) * | 2005-10-21 | 2007-04-26 | Alan Wirbisky | Compact apparatus, compositions and methods for purifying nucleic acids |
US8962262B2 (en) * | 2006-05-11 | 2015-02-24 | Arbor Vita Corporation | Method of protein extraction from cells |
US9207240B2 (en) | 2007-11-14 | 2015-12-08 | Arbor Vita Corporation | Method of efficient extraction of protein from cells |
GB201217405D0 (en) | 2012-09-28 | 2012-11-14 | Fermentas Uab | Protein removal agent |
US11377470B2 (en) | 2013-03-15 | 2022-07-05 | Modernatx, Inc. | Ribonucleic acid purification |
CN110229225B (en) * | 2019-06-12 | 2021-07-20 | 山东师范大学 | Preparation method of sulfur-rich and/or cesium-rich protein in antarctic krill |
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US20050032105A1 (en) * | 2001-10-12 | 2005-02-10 | Bair Robert Jackson | Compositions and methods for using a solid support to purify DNA |
US7148343B2 (en) * | 2001-10-12 | 2006-12-12 | Gentra Systems, Inc. | Compositions and methods for using a solid support to purify RNA |
-
2004
- 2004-12-15 AT AT04814250T patent/ATE466080T1/en not_active IP Right Cessation
- 2004-12-15 EP EP04814250A patent/EP1694692B1/en active Active
- 2004-12-15 CA CA2549985A patent/CA2549985C/en active Active
- 2004-12-15 US US11/012,527 patent/US7115719B2/en active Active
- 2004-12-15 JP JP2006545361A patent/JP4435787B2/en active Active
- 2004-12-15 DE DE602004026912T patent/DE602004026912D1/en active Active
- 2004-12-15 WO PCT/US2004/042044 patent/WO2005058933A1/en not_active Application Discontinuation
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Cited By (28)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US7776616B2 (en) | 1997-09-17 | 2010-08-17 | Qiagen North American Holdings, Inc. | Apparatuses and methods for isolating nucleic acid |
EP1727900A4 (en) * | 2004-03-26 | 2008-05-28 | Fujifilm Corp | Method for selectively separating and purifying rna and method for separating and purifying nucleic acid |
US7824855B2 (en) | 2004-03-26 | 2010-11-02 | Fujifilm Corporation | Method for selectively separating and purifying RNA and method for separating and purifying nucleic acid |
EP1727900A1 (en) * | 2004-03-26 | 2006-12-06 | Fuji Photo Film Co., Ltd. | Method for selectively separating and purifying rna and method for separating and purifying nucleic acid |
US10947527B2 (en) | 2004-11-05 | 2021-03-16 | Qiagen North American Holdings, Inc | Compositions and methods for purifying nucleic acids from stabilization reagents |
EP1960521B1 (en) * | 2005-12-07 | 2011-09-21 | AJ Innuscreen GmbH | Method and test kit for the separation, purification and recovery of long- and short-chain nucleic acids |
JP2007202514A (en) * | 2006-02-03 | 2007-08-16 | Fujifilm Corp | Method for separating and purifying nucleic acid |
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WO2009121208A1 (en) * | 2008-04-02 | 2009-10-08 | 芮宝生医股份有限公司 | Reagents, chaotropic agents, kits and methods for isolating nucleic acids based on magnetic cellulose materials |
US11820977B2 (en) | 2013-03-14 | 2023-11-21 | Translate Bio, Inc. | Methods for purification of messenger RNA |
US11692189B2 (en) | 2013-03-14 | 2023-07-04 | Translate Bio, Inc. | Methods for purification of messenger RNA |
US10876104B2 (en) | 2013-03-14 | 2020-12-29 | Translate Bio, Inc. | Methods for purification of messenger RNA |
US10590161B2 (en) | 2013-03-15 | 2020-03-17 | Modernatx, Inc. | Ion exchange purification of mRNA |
US10138507B2 (en) | 2013-03-15 | 2018-11-27 | Modernatx, Inc. | Manufacturing methods for production of RNA transcripts |
US10077439B2 (en) | 2013-03-15 | 2018-09-18 | Modernatx, Inc. | Removal of DNA fragments in mRNA production process |
WO2014144767A1 (en) * | 2013-03-15 | 2014-09-18 | Moderna Therapeutics, Inc. | Ion exchange purification of mrna |
US10155785B2 (en) | 2014-04-25 | 2018-12-18 | Translate Bio, Inc. | Methods for purification of messenger RNA |
US11884692B2 (en) | 2014-04-25 | 2024-01-30 | Translate Bio, Inc. | Methods for purification of messenger RNA |
US11059841B2 (en) | 2014-04-25 | 2021-07-13 | Translate Bio, Inc. | Methods for purification of messenger RNA |
US11268157B2 (en) | 2015-05-08 | 2022-03-08 | Curevac Real Estate Gmbh | Method for producing RNA |
US11661634B2 (en) | 2015-05-08 | 2023-05-30 | CureVac Manufacturing GmbH | Method for producing RNA |
US10017826B2 (en) | 2015-05-08 | 2018-07-10 | Curevac Ag | Method for producing RNA |
US10711315B2 (en) | 2015-05-08 | 2020-07-14 | Curevac Real Estate Gmbh | Method for producing RNA |
US11667910B2 (en) | 2015-05-29 | 2023-06-06 | CureVac Manufacturing GmbH | Method for producing and purifying RNA, comprising at least one step of tangential flow filtration |
US11760992B2 (en) | 2015-05-29 | 2023-09-19 | CureVac Manufacturing GmbH | Method for producing and purifying RNA, comprising at least one step of tangential flow filtration |
US11834651B2 (en) | 2015-05-29 | 2023-12-05 | CureVac Manufacturing GmbH | Method for producing and purifying RNA, comprising at least one step of tangential flow filtration |
US11174500B2 (en) | 2018-08-24 | 2021-11-16 | Translate Bio, Inc. | Methods for purification of messenger RNA |
Also Published As
Publication number | Publication date |
---|---|
US7115719B2 (en) | 2006-10-03 |
US20050171333A1 (en) | 2005-08-04 |
JP2007515959A (en) | 2007-06-21 |
EP1694692B1 (en) | 2010-04-28 |
DE602004026912D1 (en) | 2010-06-10 |
CA2549985A1 (en) | 2005-06-30 |
CA2549985C (en) | 2011-05-24 |
EP1694692A1 (en) | 2006-08-30 |
ATE466080T1 (en) | 2010-05-15 |
JP4435787B2 (en) | 2010-03-24 |
EP1694692A4 (en) | 2008-07-23 |
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