EP1343877A2 - Stable composition comprising a nuclease and a phosphatase - Google Patents
Stable composition comprising a nuclease and a phosphataseInfo
- Publication number
- EP1343877A2 EP1343877A2 EP01924218A EP01924218A EP1343877A2 EP 1343877 A2 EP1343877 A2 EP 1343877A2 EP 01924218 A EP01924218 A EP 01924218A EP 01924218 A EP01924218 A EP 01924218A EP 1343877 A2 EP1343877 A2 EP 1343877A2
- Authority
- EP
- European Patent Office
- Prior art keywords
- composition
- phosphatase
- nuclease
- composition according
- units
- Prior art date
- Legal status (The legal status 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 status listed.)
- Ceased
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- 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
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
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- 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
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
- C12N9/22—Ribonucleases RNAses, DNAses
Definitions
- the invention relates to the field of processing DNA, specifically including amplified DNA, to remove residual primers or other unwanted single-stranded DNA and nucleotide t ⁇ phosphates prior to performing other operations, such as, but not limited to, DNA sequencing, SNP analysis, or gene expression analysis.
- Exonuclease I digests single-stran ⁇ ed DNA in a 3' - ⁇ 5' direction producing 5' mononucleotides.
- This enzyme is particularly useful m preparing amplified DNA products, such as PCR products, for sequencing. It degrades residual primers from the amplification reaction that would otherwise be carried over into the sequencing reaction.
- 5,741,676 and 5,756,285 generally disclose methods for DNA sequencing via amplification, both of which are hereby incorporated herein by reference. (See also R.L. Olsen et al., Comp. Biochem. Physiol., vol. 99B, No. 4, pp. 755-761 (1991) ) .
- Amplification primers carried over into a sequencing reaction could act as sequencing primers and generate sequencing reaction products, thereby creating a oac ground of secondary sequences which would obscure or interfere with observing the desired sequence.
- concentration and specific activity (purity) of commercially available Exonuclease I may vary over a wide range.
- the enzyme is manufactured to a specific activity between 50,000 and 150,000 units of enzyme per mg and supplied for the purpose of processing amplified DNA at a concentration around 10 units per microliter. Enzyme with either higher or lower specific activity and either more or less concentrated could be employed m the described applications by suitable alterations m the applied protocol, such as adding less or more volume (or amount) of enzyme, respectively.
- the storage buffer of commercially available Exonuclease I is: 20 ⁇ iM Tris-HCl, pH 7.5; 0.5 itiM EDTA; 5 M 2-mercaptoethanol; 50 vol.° glycerol, made up m water (manor manufacturer and supplier, USB Corporation, Cleveland, Ohio, USA) .
- Alkaline Phosphatases as exemplified by Shrimp Alkaline Phosphatase (SAP) and Calf Intestinal Alkaline Phosphatase (CIP) , catalyze the hydrolysis of 5' -phosphate residues from DNA, RNA, and ⁇ bo- and deoxyribonucleoside triphosphates (dNTPs or nucleotide triphosphates).
- SAP is particularly useful m preparing amplified products, such as PCR products, for sequencing because it can readily be inactivated by heat prior to performing a sequencing reaction. SAP degrades residual dNTPs from the amplification reaction.
- dNTPs are carried over from the amplification reaction to the sequencing reaction, they add to, and thereby alter, the concentration of dNTPs m the sequencing reaction m an mdeterminant and non-reproducible fashion. Since, within narrow limits, high quality sequencing requires specific ratios between the sequencing reaction dNTPs and ddNTPs, an alteration m the concentration of dNTPs may result m faint sequencing reaction signals.
- the sole manufacturer of SAP has produced enzyme with a wide range of specific activities and concentrations. Examples include batches of enzyme with concentrations ranging from 4.2 units/ ⁇ l to 13.9 units/ ⁇ l with specific activities not being reported.
- Enzyme with either higher or lower specific activity and either more or less concentrated could be employed m the described applications by suitable alterations m the applied protocol such as adding less or more volume (or amount) of enzyme, respectively.
- the storage buffer of commercially available Shrimp Alkaline Phosphatase, the preferred enzyme for the above described application is: 25 inM Tris-HCl, pH 7.5; 1 mM MgCl ⁇ ; 0.1 irtM ZnCl 2 ; 50 vol.% glycerol, made up in water (available from USB Corporation, Cleveland, Ohio, USA) .
- Exo I and SAP are frequently used to process PCR reaction products.
- each enzyme is supplied in its own storage buffer as described above.
- EDTA is a chelating agent that reacts strongly with Mg 2+ and Zn ⁇ + ions. When mixed together such that the EDTA is in molar excess, the EDTA effectively sequesters Mg 2+ and Zn 2+ ions thereby preventing these ions from interacting with any protein (s) present in the solution.
- alkaline phosphatases are considered to be multimeric, metallo-enzymes that require a divalent ion, frequently Zn ⁇ + , for structural stability and activity. Consequently, there is a need in the art for a stable composition comprising both enzymes in a single delivery vehicle. Preferably, such a stable composition will enjoy a long shelf life, each enzyme retaining a significant proportion of its original functional activity over time.
- a composition comprising a nuclease and a phosphatase is provided.
- the composition is substantially free from the presence of amplified deoxyribonucleic acid.
- the phosphatase in the composition retains at least 50% of its functional activity when the composition is stored at 4°C for 24 hours.
- a method of degrading preselected nucleic acids present in a sample of material is also provided. The method comprises the step of contacting the sample with a composition comprising a nuclease and a phosphatase.
- a composition with 50% v/v of glycerol is composed of a volume of glycerol equal to one half (or 50%) of the total volume of the composition including all of its components (including glycerol and water if present) .
- concentrations reported in molarity (M) are based upon the total volume of the composition including all of its components.
- one unit of nuclease (e.g. Exo I) enzyme is that amount of nuclease enzyme required to catalyze the release of 10 nmol of acid-soluble nucleotide from denatured DNA in 30 minutes at 37 °C under standard conditions.
- one unit of phosphatase e.g.
- SAP SAP enzyme
- functional activity generally refers to the ability of an enzyme to perform its designated function as described below.
- nuclease e.g. Exo I
- Exo I is qualitatively defined m terms of the ability of nuclease enzyme to degrade residual PCR primers from PCR amplified DNA to a level low enough so as not to materially interfere with subsequent sequencing reactions or other applications. The functional activity of nuclease is measured for Exo I using the following methodology.
- Exo I as commercially supplied by USB Corporation for this application, can be used between 0.5 and 20 units, preferably 1-15 units, more preferably at about 10 units per 5 ⁇ l reaction product m standard pre-sequencmg processing of PCR amplification product.
- Exo I composition containing 10 units Exo I/ ⁇ l is prepared at time zero, and a serial dilution performed, such that the concentration of enzyme in each successive dilution is one half that of the prior dilution, for a total of preferably 5 dilutions plus the original undiluted composition. This results in the following: original undiluted composition, one half dilution, one quarter dilution, one eighth dilution, one sixteenth dilution, and one thirty-second dilution.
- the enzyme equivalents per microliter of Exo I composition in each respective dilution are: 10 units Exo I; 5 units Exo I; 2.5 units Exo I, 1.25 units Exo I, 0.625 units Exo I; and 0.3125 units Exo I; corresponding to the undiluted composition, as well as dilutions equal to one half, one fourth, one eighth, one sixteenth, and one thirty-second the concentration of the undiluted composition.
- each of the above is separately delivered to a separate 5 ⁇ l sample of a control PCR reaction product (which has been pretreated or is being co-treated to materially degrade the dNTPs) containing residual DNA primers to be degraded prior to sequencing, and the enzyme is permitted to degrade the residual primers.
- the sequencing is then performed and the sequence ladders (six in this example) compared. In looking at the sequence ladders or lanes, the first dilution where the sequencing ladder exhibits material secondary and/or multiple lane signals compared to the primary sequencing signal indicates that the enzyme activity dropped off at that dilution. This is referred to as the "drop-off dilution".
- This is used as a measuring stick or baseline for determining, at a subsequent point in time, the half-life and functional activity of the enzyme.
- a similar serial dilution analysis is performed on a portion of the original stored composition, and the "drop- off dilution" is again ascertained.
- the first time that the "drop-off dilution" shifts from one dilution (for example, the one sixteenth dilution) to the prior dilution (for example, the one eighth dilution) indicates the point in time that the half-life of the nuclease enzyme has been reached.
- the drop-off dilution is the one thirty-second dilution.
- the composition is then stored at 4°C for a period of time, say one week.
- the stored composition is again subjected to serial dilution analysis, and the drop-off dilution remains the one thirty- second dilution.
- Serial dilution analyses are subsequently performed at 2, 3, 4, 5, etc., weeks, and it is found at the 5 th week test that, for the first time, the drop-off dilution is the one sixteenth dilution. This indicates that the half- life point has been reached. In this example, it can be seen that the half-life point was reached between the fourth and fifth weeks.
- the nuclease enzyme in the composition retained at least 50% of its functional activity when the composition was stored for four weeks at 4°C.
- the functional activity of phosphatase e.g. SAP
- SAP phosphatase
- the functional activity of phosphatase is measured for SAP using the following methodology. 1 ⁇ l of a solution containing SAP is added to 5 ⁇ l of PCR amplified DNA and the mixture incubated at 37 °C for 15 minutes. The reaction is terminated by heating to 80°C for 15 minutes.
- the treated DNA is then used as template m a standard sequencing reaction, such as the USB T"-Sequenase V2.0 PCR Product Sequencing Kit, and the quality of the sequencing ladder examined to determine the effectiveness of degrading residual nucleotide triphosphates from the amplified DNA. If residual nucleoti ⁇ e triphosphates m PCR amplified DNA are not effectively degraded, the nucleotide triphosphates from the PCR reaction will alter the ratio of dNTPs/ddNTPs in the sequencing reaction causing faint signals.
- a standard sequencing reaction such as the USB T"-Sequenase V2.0 PCR Product Sequencing Kit
- Independently formulated SAP as commercially supplied by USB Corporation for this application, can be used to degrade residual nucleotide triphosphates m PCR amplified DNA between 0.1 and 5 units, preferably 1-3 units, more preferably at about 2 units per 5 ⁇ l reaction product m standard pre-sequencmg processing of PCR amplification product.
- the functional activity and half-life of SAP and other phosphatases of the invention are ascertained v__a periodic serial dilution analyses similarly as explained above with respect to Exo I.
- An original SAP composition containing 2 units SAP per ⁇ l is prepared, and 1 ⁇ l of the original undiluted SAP composition and 5 serial dilutions thereof are delivered separately to separate 5 ⁇ l samples of a control PCR reaction product (preferably having been pretreated or being co-treated to degrade residual primers) having residual nucleotide triphosphates to be cleaned up, and the enzyme is permitted to degrade the nucleotide triphosphates.
- the sequencing is then performed and the sequence ladders compared as before. In looking at the sequence ladders or lanes, the first dilution where the first 50 bases of a DNA sequencing ladder having more than 200 discernable bases are materially fainter than m the prior dilution indicates that the enzyme activity dropped off at that dilution.
- the drop-off dilution is used as a measuring stick or baseline for determining, at subsequent points m time, the half-life and functional activity of the enzyme.
- a similar serial dilution analysis is performed on a portion of the original stored composition, and the "drop- off dilution” is again ascertained.
- Half-life for SAP is then determined similarly as explained above with respect to Exo I. For example, an original SAP composition containing 2 units SAP per ⁇ l is prepared and subject to a serial dilution analysis as described above.
- the drop-off dilution at time zero is the one thirty-second dilution.
- the composition is then stored at 4°C for a period of time, say one week.
- the stored composition is then subjected to another serial dilution analysis, and the drop-off dilution remains the one thirty-second dilution.
- Serial dilution analyses are subsequently performed at 2, 3, 4, 5, etc., weeks, and it is found at the 5 th week test that, for the first time, the drop- off dilution is the one sixteenth dilution.
- the half-life point was reached between the fourth and fifth weeks.
- the phosphatase enzyme m the composition retained at least 50% of its functional activity when the composition was stored for four weeks at 4°C.
- the present invention relates to a single composition comprising both a nuclease and a phosphatase, wherein less than 50%, preferably less than 40%, preferably less than 30%, preferably less than 20%, preferably less than 10%, of the functional activity of each and/or either enzyme is lost per 24 hours, more preferably per week, even more preferably per month, and most preferably per 4 months, when held or stored under a specified condition such as -20°C, 0°C, +4°C, or room temperature (e.g. +20°C).
- the phosphatase in the composition preferably retains at least 50% of its functional activity when said composition is stored at 4°C for 24, more preferably 36, more preferably 48, more preferably 60, more preferably 72, more preferably 96, hours.
- the nuclease in the composition preferably retains at least 50% of its functional activity when said composition is stored at 4°C for 2, more preferably 3, more preferably 5, more preferably 7, more preferably 9, more preferably 12, more preferably 14, days.
- the invented composition is preferably substantially free from the presence of deoxyribonucleic acid, nucleic acid, amplified DNA, nucleotide triphosphates, oligonucleotides, and primers, each of which could interfere with the composition's performance.
- the nuclease is heat-labile, preferably single-stranded exonuclease, preferably Exonuclease 7 or RecJ, most preferably Exo I
- the phosphatase is preferably heat- labile, preferably eukaryotic phosphatase, preferably bacterial or animal phosphatase, preferably mammal phosphatase, most preferably SAP.
- the invented composition preferably is formulated in such a manner that when an aliquot of 2 ⁇ l of the composition is contacted with 5 ⁇ l of PCR reaction product (DNA that was amplified by standard PCR techniques), the residual primers and nucleotide triphosphates are effectively inactivated or degraded by being decreased to a level that allows effective sequencing of the amplified product.
- the amounts and concentrations of the Exo I, SAP and other materials may vary depending upon the specific nature and amount of the amplified DNA product, the nature and amount of residual primers and nucleotide triphosphates, the time and temperature of the processing reaction, and the sequencing method used.
- Embodiments of the invention also allow for adding different volumes or proportions of the combined composition as needed to achieve the desired result.
- compositions containing nuclease, such as Exo I, and phosphatase, such as SAP to be dehydrated or dried (or optionally lyophilized) , thus comprising at most 10 wt . % water, and these concentrated or dried forms to be contacted with the amplified DNA.
- the invention provides a nuclease and a phosphatase in a single composition.
- the composition can be used for degrading residual materials present in the product of a nucleic acid synthesis reaction, examples of which are referenced or described in this paragraph.
- the method involves contacting (for example, mixing) the reaction product with the composition.
- the composition can be used for cleaning up or degrading residual primers and residual nucleotide triphosphates, preferably after a DNA or RNA amplification reaction, preferably a PCR or RT-PCR amplification reaction, alternatively an isothermal amplification reaction.
- the composition can also be used for cleaning up a nucleic acid (preferably DNA) replication reaction, such as primer- initiated RNA or DNA synthesis.
- the cleaned-up reaction product can be used in subsequent analyses, such as DNA sequencing, less preferably SNP (Single Nucleotide Polymorphism) analysis (which is a way of determining single nucleotide differences), other genetic analyses (including gene expression) or other analyses of nucleic acids where cleanup of residual primers, residual oligonucleotides and/or residual nucleotide triphosphates is useful, such as analysis of multiple base additions, deletions or differences.
- SNP Single Nucleotide Polymorphism
- the invented composition can also be used, with or without additional nucleases and/or phosphatases, to act as a selective and/or all-purpose clean-up composition to clean up samples other than amplification reaction products, such as a biological sample such as biopsy materials, blood samples, bodily fluids, or intermediates used in the production of biological materials.
- a biological sample such as biopsy materials, blood samples, bodily fluids, or intermediates used in the production of biological materials.
- the composition containing a nuclease and a phosphatase would degrade preselected nucleic acids present in the sample of material.
- the sample could be material, such as biopsy material, isolated from biological material, such as a human body.
- the referenced stability generally relates to compositions held in either liquid or dried states. However, it is recognized that combinations of Exo I and SAP can be stored frozen.
- compositions of Exo I and SAP could be held with potentially little reduction in functional activity or performance for extended periods of time such as at least 6, 12, 24, 36, 60 or 100 months.
- the invented composition retains at least 10, 20, 30, 40, 50, 60, 70, 80 and/or 90 % of its functional activity for each enzyme following storage of the composition for 24 hours, or 2, 3, 4, 5, 8, 10, 15, 20, 30, 40, 60, 80, 100, 120, 150, 180, 210, 240, 300, 360, 500, 1000, 1500, 2000 and/or 3000 days at 25 C, 20°C, 18°C, 10°C, 4 U C, 0 ⁇ C, -10 U C, -20°C, -30 U C, -40°C, -60°C, -80°C, -100°C, -150°C or -190 U C.
- the invented compositions are packaged, stored, shipped and used as known in the art.
- the only necessary components of the invented composition are the enzymes, that is, the nuclease and the phosphatase.
- the other components described herein are preferred but are optional.
- the nuclease is preferably Exonuclease I (Exo I) and the phosphatase is preferably alkaline phosphatase, preferably Shrimp Alkaline Phosphatase (SAP) as indicated above.
- the combination of enzymes can be supplied in dried form or, more preferably, m a liquid, preferably m an aqueous solution. Preferred aqueous solutions are described herein.
- the enzymes can be supplied m more concentrated solutions, such as solutions (with or without the optional components) which are at least 2, 3, 4, 5, 6, 8, 10, 15, 20, 30, 50, 80, 100, 150, 200, 300, 500, 800, 1,000, 2,000, 5,000, 8,000, or 10,000 times more concentrated than the solutions described herein, or concentrated all the way to dryness. Diluted solutions can also be provided.
- any preferred or less preferred concentration or range of any component can be combined with any preferred or less preferred concentration or range of any of the other component or components; it is not required or necessary that all or any of the components or concentrations or ranges be that which is most preferred.
- the composition is a liquid, preferably aqueous, combination of a nuclease and a phosphatase (preferably an alkaline phosphatase) , preferably Exo I and SAP, where the Exo I to SAP unit ratio is between 1:5000 and 5000:1, more preferably between 1:500 and 500:1, even more preferably between 1:50 and 50:1 and most preferably between 1:10 and 10:1 with a total protein concentration ranging from 1 ⁇ g/ml to 200 mg/ml, more preferably 10 ⁇ g/ml to 100 mg/ml, even more preferably 100 ⁇ g/ml to 50 mg/ml and most preferably between 1.0 mg/ml and 10 mg/ml.
- a nuclease and a phosphatase preferably an alkaline phosphatase
- SAP preferably Exo I and SAP
- the units of Exo I contacted with 5 ⁇ l PCR amplified DNA could range from 0.01 to 100 units of Exo I, more preferably 0.1 to 30 units of Exo I, even more preferably 1 to 15 units of Exo I and most preferably 10 ⁇ 4 units of Exo I
- the 5 ⁇ l PCR amplification reaction product is also preferably contacted with 0.01 to 100 units of SAP, more preferably 0.1 to 10 units of SAP, even more preferably 0.5 to 5 units of SAP and most preferably 2 ⁇ 1 units of SAP.
- other alkaline phosphatates such as calf intestinal alkaline phosphatase, may be used in place of the SAP.
- the concentration of nuclease in the invented composition is preferably at least 0.01, 0.1, 1, 2, or 5 units of nuclease enzyme per microliter.
- the concentration of phosphatase in the invented composition is preferably at least 0.01, 0.1, 1, 2, or 5 units of phosphatase enzyme per microliter.
- the pH is between 4.0 and 12.0, more preferably between pH 6.0 and 10.0, more preferably between 7.0 and 9.0, more preferably less than 8, more preferably between 7 and 8, and most preferably pH 7.5 ⁇ 0.2 or pH 7.5 ⁇ 0.3, preferably controlled by a buffer.
- the invented composition may optionally and preferably contain a buffer at a concentration of zero to 250 mM, more preferably between 5 mM to 100 mM, even more preferably between 15 mM to 50 mM and most preferably 25 ⁇ 5 mM, preferably of Tris-HCl, preferably at pH 7.5 to pH 8.5 or the pH ranges mentioned above.
- Other buffers may be used such as, but not limited to: organic buffers such as MOPS, HEPES, TRICINE, etc., or inorganic buffers such as Phosphate or Acetate. Buffers or other agents may be added to control the pH of the solution thereby increasing the stability of the enzymes.
- the invented composition may optionally and preferably contain a reducing agent such as but not limited to: dithiotreitol (DTT) or 2-mercaptoethanol; preferably zero to 100 mM, more preferably 0.1 mM to 50 mM, even more preferably 0.5 to 10 mM and most preferably 1.0 ⁇ 0.2 mM.
- a reducing agent such as but not limited to: dithiotreitol (DTT) or 2-mercaptoethanol
- DTT dithiotreitol
- 2-mercaptoethanol preferably zero to 100 mM, more preferably 0.1 mM to 50 mM, even more preferably 0.5 to 10 mM and most preferably 1.0 ⁇ 0.2 mM.
- Reducing agents may be added to limit enzyme oxidation that might adversely affect stability of the enzymes.
- the invented composition may optionally and preferably contain onovalent ions such as, but not limited to: Na + , K , Li + , Cl ⁇ , Br " or acetate (HCO ) at a concentration of zero to 500 mM, more preferably 0.5 mM to 100 mM, even more preferably 1 mM to 50 mM and most preferably 1 to 10 mM.
- onovalent ions such as, but not limited to: Na + , K , Li + , Cl ⁇ , Br " or acetate (HCO ) at a concentration of zero to 500 mM, more preferably 0.5 mM to 100 mM, even more preferably 1 mM to 50 mM and most preferably 1 to 10 mM.
- HCO acetate
- the invented composition may optionally and preferably contain a complexing or chelating agent such as, but not limited to, Na 2 -EDTA or Na 2 -EGTA at a concentration of zero to 100 mM, more preferably 0.05 to 10 mM, even more preferably 0.1 to 2 mM, and most preferably 0.5 ⁇ 0.1 mM.
- a complexing or chelating agent such as, but not limited to, Na 2 -EDTA or Na 2 -EGTA at a concentration of zero to 100 mM, more preferably 0.05 to 10 mM, even more preferably 0.1 to 2 mM, and most preferably 0.5 ⁇ 0.1 mM.
- Chelating agents are frequently added to protein solutions to sequester metal ions which if present can catalyze changes in amino acid side chain chemistry and under certain conditions cause breaks in the amino acid backbone of enzymes, thereby decreasing activity.
- the invented composition may optionally contain an amino acid based carrier or stabilizer such as, but not limited to, bovine serum albumin and Poly L-lysine, preferably at a concentration between zero and 100 mg/ml, more preferably between 0.01 and 10 mg/ml and most preferably between 0.1 and 1.0 mg/ml.
- the invented composition may optionally contain divalent ions such as but not limited to: Zn + , Mg _+ , Co + , Mn 2+ and/or Ca"" + , preferably at a concentration between zero and 200 mM, more preferably between zero and 20 mM, more preferably between 0.0001 mM and 5 mM and most preferably 0.002 to 1.0 mM.
- Divalent ions are preferred or required for effective enzyme activity of some proteins, such as phosphatases. Trace amounts of divalent ions may be present as a result of the addition of other substances to the composition; the normal composition of SAP contains both Zn + and Mg ⁇ + which may accompany the enzyme into the composition.
- the invented composition may optionally contain detergents (singly or in combination) such as, but not limited to, non-ionic, ionic or zwitterionic detergents added to stabilize the enzymes or enhance performance.
- detergents such as, but not limited to, non-ionic, ionic or zwitterionic detergents added to stabilize the enzymes or enhance performance.
- Nonidet P40, Triton X100 or Tween 20 between zero and 20% v/v, more preferably between 0.01% and 5% v/v, and most preferably between 0.1% and 1.0% v/v.
- the invented composition may optionally contain other chemicals added that enhance performance such as, but not limited to, DMSO between zero and 50% v/v, more preferably between 0.001% and 10% v/v, most preferably between 0.01% and 1% v/v.
- the invented composition may optionally contain a dextran such as Dextran T-10 or Dextran T500 or other polysaccharide between zero and 50% v/v, more preferably between 0.1% and 10% v/v and most preferably between 1% and 5% v/v.
- the invented composition may optionally and preferably contain an enzyme stabilizer or a material that inhibits ice formation such as, but not limited to, glycerol, ethylene glycol or glycine, preferably glycerol, preferably at a concentration of zero to 99% v/v, more preferably 1% to 75% v/v, more preferably 5% to 65% v/v, more preferably 20% to 60% v/v, more preferably 35% to 58% v/v, and most preferably 50 ⁇ 5% v/v.
- the invented composition may optionally contain mono- or disaccharide such as glucose or maltose that may stabilize the enzymes or facilitate the composition of a dry embodiment.
- the mass of the mono- or disaccharide is preferably at least zero, 0.1, 1, 10, 100, 1000 or 10,000, or not more than 10 or 100 or 1000 or 10,000, times the mass of the protein m the composition.
- compositions D and E are described below as Compositions D and E.
- Composition D is preferred for manual pipetting operations, and composition E is preferred for automated pipetting operations.
- composition D is used, preferably 2 ⁇ l of composition D are combined with 5 ⁇ l of PCR reaction product to effectively degrade residual primers and nucleotide triphosphates prior to sequencing.
- composition E preferably 5 ⁇ l of composition E are combined with 5-25 ⁇ l, preferably 5 ⁇ l, of PCR reaction product to effectively degrade residual primers and nucleotide triphosphates prior to sequencing or other analyses.
- composition D or E it is preferred that 10 units of Exo I and 2 units of SAP are delivered to 5 ⁇ l of product containing residual primers and/or nucleotide triphosphates to be degraded.
- compositions A through E 5 separate nuclease/phosphatase compositions were prepared, and are generally referred to herein as Compositions A through E.
- the compositions and component concentrations of each composition are provided below.
- Composition A was prepared as an aqueous composition with the following components: 10 units/ ⁇ l of Exonuclease I; 2 units/ ⁇ l of Shrimp Alkaline Phosphatase; 25 mM Tris-HCl, pH 7.5; 0.5 mM Na_-EDTA; 1 mM DTT; 50% v/v glycerol, made up m water.
- Exo I and SAP Concentrated stocks of Exo I and SAP were dialyzed against 25 mM Tris-HCl, pH 7.5; 0.5 mM Na_-EDTA; 1 mM DTT; 50% v/v glycerol. Following dialysis the enzymes were combined in Composition A so that each microliter of Composition A contained 10 units of Exo I and 2 units of SAP. Enzyme activity assays as well as enzyme functional activity were measured, as indicated in table 1, after the composition was stored at -20°C, 4°C and +25°C for various lengths of time.
- Composition B was prepared as an aqueous composition with the following components: 10 units/ ⁇ l of Exonuclease I; 2 units/ ⁇ l of Shrimp Alkaline Phosphatase; 25 mM Tris-HCl, pH 7.5; 100 ⁇ g/ml bovine serum albumin; 1 mM DTT; 1 mM MgCl?; 0.1 mM ZnCl 2 ; 50% v/v glycerol, made up in water.
- Exo I and SAP Concentrated stocks of Exo I and SAP were dialyzed against 25 mM Tris-HCl, pH 7.5; 100 ⁇ g/ml bovine serum albumin; 1 mM DTT; 1 mM MgCl 2 ; 0.1 mM ZnCl 2 ; 50% v/v glycerol. Following dialysis the enzymes were combined in Composition B so that each microliter of Composition B contained 10 units of Exo I and 2 units of SAP. Enzyme functional activity was measured, as indicated in table 1, after the composition was stored at -20°C, 4°C and +25°C for various lengths of time.
- Composition C was prepared as an aqueous composition with the following components: 10 units/ ⁇ l of Exonuclease I; 2 units/ ⁇ l of Shrimp Alkaline Phosphatase; formulated into 50 mM Tris-HCl, pH 8.3; 0.5 mM Na-EDTA; 1 mM DTT; 0.5% v/v Tween 20; 0.5% v/v Nonidet P-40, 50% v/v glycerol, made up in water.
- the composition was made by mixing the appropriate amount of Exo I and SAP, in their commercially available storage buffers, into Composition C. This composition thus contained small amounts of MgCl ⁇ and ZnCl.> derived from the commercial SAP composition.
- Composition D was prepared as an aqueous composition with the following components: 5 units/ ⁇ l of Exonuclease I; 1 unit/ ⁇ l of Shrimp Alkaline Phosphatase; formulated into 25 mM Tris-HCl, pH 7.5; 0.5 mM Na : -EDTA; lmM DTT; 50% v/v glycerol. This composition was made by mixing the appropriate amount of Exo I and SAP, in their commercially available storage buffers, into Composition D.
- Composition D thus contains traces of MgCl and ZnCl derived from the commercial SAP composition, and 2-mercaptoethanol derived from the Exo I composition.
- a working volume of 2 ⁇ l of this enzyme mixture was used. Enzyme functional activity was measured, as indicated in table 1, after the composition was stored at -80°C, -20°C, 4°C, and 25°C for various lengths of time. A freeze and thaw experiment was also performed.
- Composition E was prepared as an aqueous composition with the following components: 2 units/ ⁇ l of Exonuclease I; 0.4 units/ ⁇ l of Shrimp Alkaline Phosphatase; formulated into 25 mM Tris-HCl, pH 7.5; 0.5 mM Na ⁇ -EDTA; 1 mM DTT; 50% v/v glycerol.
- This composition was made by mixing the appropriate amount of Exo I and SAP, in their commercially available storage buffers, into Composition E.
- Composition E thus contains traces of MgCl 2 and ZnCl derived from the commercial SAP composition, and 2-mercaptoethanol derived from the Exo I composition.
- a working volume of 5 ⁇ l for this enzyme mixture is a convenient volume for addition to PCR reaction mixtures by robotic pipetters .
- Enzyme functional activity was measured, as indicated in table 1, after the composition was stored at -20°C for various lengths of time.
- the functional activity of each of the above nuclease/phosphatase compositions was determined at the various stated temperatures and after the stated elapsed times as described above and further as described below.
- a sample of each composition was removed as appropriate and a serial 1:1 dilution made into the respective composition, such that the concentration of enzyme in each successive dilution was one half that of the prior dilution.
- compositions A-C presuming no change in activity, these enzyme equivalents per volume addition to the PCR reaction product (per ⁇ l of the enzyme composition) were: 10 units Exo I with 2 units SAP; 5 units Exo I with 1 unit SAP; 2.5 units Exo I with 0.5 units SAP; 1.25 units Exo I with 0.25 units SAP; 0.625 units Exo I with 0.125 units SAP; and 0.3125 units Exo I with 0.0625 units SAP.
- These amounts thus represented the respective undiluted compositions, as well as dilute compositions diluted to one half, one fourth, one eighth, one sixteenth, and one thirty- second the concentration of the respective undiluted compositions.
- These serial dilutions resulted in concentration of enzyme that paralleled those made with untreated Exo I and SAP stock enzyme.
- the activity half-life as expressed in table 1 is that duration of storage required to observe a 50 reduction in functional activity of either the Exo I or the SAP in the composition.
- EXAMPLE 2 STABILITY AT -20°C OF EXONUCLEASE I AND SHRIMP ALKALINE PHOSPHATASE ENZYMES IN A COMBINED COMPOSITION
- Compositions A, B and D showed significant retention in functional activity of either the Exonuclease I or shrimp alkaline phosphatase as compared to their respective control enzymes. Even more unexpectedly, upon formulation over a 100. gain in SAP functional activity was observed in the test of Compositions A and D, the compositions containing an excess of EDTA. In this test when only 0.25 units of commercially formulated SAP (a 1/8 dilution) were used to react amplified PCR DNA, the bottom of the DNA sequence ladder was faint.
- composition B exhibits an unexpected retention in functional activity (see table 1), but did not exhibit the unexpected increase in activity exhibited by Compositions A and D.
- Composition E also unexpectedly exhibited significant retention in activity (see table 1) .
- EXAMPLE 3 STABILITY AT +4°C OF EXONUCLEASE I AND SHRIMP ALKALINE PHOSPHATASE ENZYMES IN A COMBINED COMPOSITION
- considerable functional activity of SAP in Composition A and Composition D was retained following storage at +4°C with less than 50% of its functional activity being lost in three days. (See table 1) .
- EXAMPLE 4 STABILITY AT +25°C OF EXONUCLEASE I AND SHRIMP ALKALINE PHOSPHATASE ENZYMES IN A COMBINED COMPOSITION
- Composition A as well as Composition D was retained following storage at +25°C with as much as 25% of the original functional activity being retained after one day of storage at +25°C. This retention of activity appears to be even greater than that reported for SAP when stored in its normal, commercially available composition ("Shrimp Alkaline Phosphatase", Monograph, Biotec-Mackzymal AS, Tromso, Norway).
- EXAMPLE 5 STABILITY AT -80 °C OF EXONUCLEASE I AND SHRIMP ALKALINE PHOSPHATASE ENZYMES IN COMBINED COMPOSITION D Upon thawing after 8 weeks of storage at -80°C, Composition D exhibited no detectable loss of functional activity of either Exonuclease I or Micromp Alkaline Phosphatase.
- combined nuclease/phosphatase compositions according to the invention can be prepared using other, less preferred components and component concentrations.
- Table 2 summarizes various components and component concentrations that can be used in the invented composition. In table 2, any preferred or less preferred or more preferred concentration or range of any component can be combined with any preferred or less preferred or more preferred concentration or range of any of the other components; it is not required or necessary that all or any of the concentrations or ranges come from the same column.
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Abstract
Description
Claims
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP14173528.2A EP2803724A1 (en) | 2000-03-21 | 2001-03-20 | Stable composition comprising a nuclease and a phosphatase |
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US19081300P | 2000-03-21 | 2000-03-21 | |
US190813P | 2000-03-21 | ||
PCT/US2001/008859 WO2001070943A2 (en) | 2000-03-21 | 2001-03-20 | Stable composition comprising a nuclease and a phosphatase |
Related Child Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP14173528.2A Division EP2803724A1 (en) | 2000-03-21 | 2001-03-20 | Stable composition comprising a nuclease and a phosphatase |
Publications (1)
Publication Number | Publication Date |
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EP1343877A2 true EP1343877A2 (en) | 2003-09-17 |
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ID=22702898
Family Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP01924218A Ceased EP1343877A2 (en) | 2000-03-21 | 2001-03-20 | Stable composition comprising a nuclease and a phosphatase |
EP14173528.2A Withdrawn EP2803724A1 (en) | 2000-03-21 | 2001-03-20 | Stable composition comprising a nuclease and a phosphatase |
Family Applications After (1)
Application Number | Title | Priority Date | Filing Date |
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EP14173528.2A Withdrawn EP2803724A1 (en) | 2000-03-21 | 2001-03-20 | Stable composition comprising a nuclease and a phosphatase |
Country Status (6)
Country | Link |
---|---|
EP (2) | EP1343877A2 (en) |
JP (1) | JP3803295B2 (en) |
AU (1) | AU2001250890A1 (en) |
CA (1) | CA2403438C (en) |
NO (1) | NO20024537L (en) |
WO (1) | WO2001070943A2 (en) |
Families Citing this family (2)
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JP4662929B2 (en) * | 2004-04-01 | 2011-03-30 | 独立行政法人理化学研究所 | Bovine major histocompatibility complex (BoLA) class IIDQA allele determination method |
DE102009012039A1 (en) | 2009-03-10 | 2010-09-16 | Qiagen Gmbh | Quantification of nucleic acids |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1993006243A1 (en) * | 1991-09-27 | 1993-04-01 | The United States Biochemical Corporation | Dna cycle sequencing |
WO2001011085A2 (en) * | 1999-08-11 | 2001-02-15 | Whitehead Institute For Biomedical Research | Bdnf polymorphism and association with bipolar disorder |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5756285A (en) | 1991-09-27 | 1998-05-26 | Amersham Life Science, Inc. | DNA cycle sequencing |
-
2001
- 2001-03-20 EP EP01924218A patent/EP1343877A2/en not_active Ceased
- 2001-03-20 CA CA002403438A patent/CA2403438C/en not_active Expired - Lifetime
- 2001-03-20 AU AU2001250890A patent/AU2001250890A1/en not_active Abandoned
- 2001-03-20 JP JP2001569326A patent/JP3803295B2/en not_active Expired - Lifetime
- 2001-03-20 EP EP14173528.2A patent/EP2803724A1/en not_active Withdrawn
- 2001-03-20 WO PCT/US2001/008859 patent/WO2001070943A2/en active Application Filing
-
2002
- 2002-09-20 NO NO20024537A patent/NO20024537L/en not_active Application Discontinuation
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1993006243A1 (en) * | 1991-09-27 | 1993-04-01 | The United States Biochemical Corporation | Dna cycle sequencing |
WO2001011085A2 (en) * | 1999-08-11 | 2001-02-15 | Whitehead Institute For Biomedical Research | Bdnf polymorphism and association with bipolar disorder |
Non-Patent Citations (2)
Title |
---|
"ExoSAP-IT Protocol", 2000, Retrieved from the Internet <URL:http://sequencingfacility.med.monash.edu.au/pdf/Exosap.pdf> [retrieved on 20080731] * |
See also references of WO0170943A3 * |
Also Published As
Publication number | Publication date |
---|---|
CA2403438A1 (en) | 2001-09-27 |
JP2004501611A (en) | 2004-01-22 |
CA2403438C (en) | 2008-10-21 |
EP2803724A1 (en) | 2014-11-19 |
WO2001070943A3 (en) | 2003-07-17 |
JP3803295B2 (en) | 2006-08-02 |
AU2001250890A1 (en) | 2001-10-03 |
WO2001070943A2 (en) | 2001-09-27 |
NO20024537L (en) | 2002-11-20 |
NO20024537D0 (en) | 2002-09-20 |
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