Stable composition comprising a nuclease and a phosphatase

Details Stable composition comprising a nuclease and a phosphatase Stable composition comprising a nuclease and a phosphatase

2001-03-20

2002-04-30

Fredman, Jeffrey (Department: 1656)

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Stablizing an enzyme by forming a mixture, an adduct or a...

C435S091200, C435S187000, C435S199000, C514S002600

Reexamination Certificate

active

06379940

ABSTRACT:

BACKGROUND OF THE INVENTION
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 triphosphates prior to performing other operations, such as, but not limited to, DNA sequencing, SNP analysis, or gene expression analysis.
Exonuclease I (Exo I) digests single-stranded DNA in a 3′→5′ direction producing 5′ mononucleotides. This enzyme is particularly useful in 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. U.S. Pat. Nos. 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 background of secondary sequences which would obscure or interfere with observing the desired sequence. Both the concentration and specific activity (purity) of commercially available Exonuclease I may vary over a wide range. Commonly 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 in the described applications by suitable alterations in 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 mM Tris-HCl, pH 7.5; 0.5 mM EDTA; 5 mM 2-mercaptoethanol; 50 vol. % glycerol, made up in water (major 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 ribo- and deoxyribonucleoside triphosphates (dNTPs or nucleotide triphosphates). SAP is particularly useful in 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. If residual dNTPs are carried over from the amplification reaction to the sequencing reaction, they add to, and thereby alter, the concentration of dNTPs in the sequencing reaction in an indeterminant and non-reproducible fashion. Since, within narrow limits, high quality sequencing requires specific ratios between the sequencing reaction dNTPs and ddNTPs, an alteration in the concentration of dNTPs may result in 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/&mgr;l to 13.9 units/&mgr;l with specific activities not being reported. Enzyme with either higher or lower specific activity and either more or less concentrated could be employed in the described applications by suitable alterations in 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 mM Tris-HCl, pH 7.5; 1 mM MgCl
2
; 0.1 mM ZnCl
2
; 50 vol. % glycerol, made up in water (available from USB Corporation, Cleveland, Ohio, USA).
Prior to sequencing or other analyses, Exo I and SAP are frequently used to process PCR reaction products. Currently each enzyme is supplied in its own storage buffer as described above. In a recommended procedure (see “PCR Product Pre-Sequencing Kit” protocol booklet, USB Corporation) one microliter of each enzyme preparation is independently added (via pipetting) to 5 microliters of PCR reaction product. In this application multiple pipetting steps potentially can introduce significant experimental error, both determinant and indeterminant, into subsequent sequencing measurements. Furthermore, the ratio of Exo I to SAP can vary significantly among subsequent experiments due to delivery of imprecise relative volumes of each of the enzyme preparations to subsequent batches of amplified DNA.
Historically, a stable composition comprising both enzymes in fixed proportion has not been commercially produced. It may have been thought that the MgCl
2
and ZnCl
2
, both present in the commercial SAP storage buffer, were incompatible with the EDTA present in the commercial Exo I storage buffer. EDTA is a chelating agent that reacts strongly with Mg
2+
and Zn
2+
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. As a class, alkaline phosphatases are considered to be multimeric, metallo-enzymes that require a divalent ion, frequently Zn
2+
, 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.
SUMMARY OF THE INVENTION
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.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION
As used herein, when a range such as 5-25 or 5 to 25 or between 5 and 25 is given, this means preferably at least 5 and, separately and independently, preferably not more than 25.
As used herein, and in the appended claims, when the concentration of a component is provided as a volume/volume percent (% v/v), this means that that component is present by volume in a proportion relative to the total volume of the composition (including all of its constituent components) equal to the stated percent for the specific component. By way of example, 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). In such a composition, concentrations reported in molarity (M) are based upon the total volume of the composition including all of its components.
As used herein, 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.
As used herein, one unit of phosphatase (e.g. SAP) enzyme is that amount of phosphatase enzyme required to catalyze the hydrolysis of 1 &mgr;mol of p-nitrophenylphosphate per minute in glycine/NaOH buffer (pH 10.4) at 37° C.
As used herein, the term “functional activity” generally refers to the ability of an enzyme to perform its designated function as described below. As used herein, the functional activity of nuclease (e.g. Exo I) is qualitatively defined in 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 ac

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