Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
2000-08-17
2001-08-28
Ketter, James (Department: 1636)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C435S320100
Reexamination Certificate
active
06281349
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a novel method for the purification of nucleic acid preparations. More specifically, the present invention provides a novel method using iodine for purifying plasmid nucleic acid suitable for several different applications in molecular biology.
BACKGROUND OF THE INVENTION
Preparation of plasmid DNA has become a central protocol in modern molecular biology. Conventional existing techniques for the isolation of plasmid DNA from cells include alkaline lysis, lithium miniprep, boiling miniprep and triton-lysozyme lysis. Such techniques have various limitations such as being time-consuming, complex or simply not suitable for large scale manufacturing processes. Also, these techniques tend to yield an impure DNA product. For example, in the alkaline lysis method, the denatured closed circular form of plasmid DNA formed during the procedure often contaminates the resultant plasmid.
Still other types of methods have also been developed for the isolation of plasmid DNA samples. For example, U.S. Pat. No. 5,561,064 describes a method for producing plasmid DNA which incorporates chromatography for purification. U.S. Pat. No. 5,660,984 describes a method and apparatus for isolating a plasmid DNA sample in which an anion-exchange resin is utilized. U.S. Pat. No. 5,707,812 describes a method for purifying plasmid DNA which also incorporates column chromatography for purification.
Commercial kits have also been specifically designed for the isolation of plasmid DNA and now represent a market worth many millions of dollars per year. For academic laboratories stressed by restricted fiscal resources these kits represent a substantial cost which must be considered against the additional time required for the older, more complex protocols. Most of these kits share a number of steps derived from the alkaline lysis protocol of Bimboim and Doly (
1
) which has subsequently been improved by the addition of a step in which the DNA is separated from other cellular components while adsorbed to a resin, (usually silica particles). This added step improves the purity of the product by removing some residual protein and most of the residual RNA.
Despite the improved quality of plasmid DNA isolated by existing commercial methods, preparations from many
E. coli
strains are contaminated by trace amounts of an extremely stable nuclease, namely Endonuclease I (Endo I) and pancreatic ribonuclease introduced in these methods for the purpose of degrading cellular RNA. Endo I, like many other nucleases, is inactive until magnesium is added to the DNA as required by most DNA metabolizing enzymes. The degradation of the plasmid that this enzymatic contamination causes a serious problem if the DNA is to be used in most molecular biology protocols. When recognized the problem may be overcome through the use of an Endo I negative
E coli
host for the plasmid or through the inclusion of additional steps such as phenol extraction step in the procedure. Such steps such are required whenever it is necessary to transcribe the plasmid since pancreatic ribonuclease, used to reduce contamination of the plasmid by cellular RNA, is sufficiently stable that it can survive the chaotropic solvents used to bind the DNA to the silica matrix.
These modifications to existing conventional protocols are inconvenient. There is no simple selection by which an Endo I deficient mutant of
E. coli
can be derived from a strain with otherwise desired genotypic characteristics. Phenol extraction is especially time consuming since it requires careful separation of the phases and subsequent complete removal of the phenol. Phenol is also hazardous.
There was therefore a need to develop a reliable, fast, efficient and economical method for the preparation of plasmid nucleic acid that obviated at least one of the limitations of the methods of the prior art.
SUMMARY OF THE INVENTION
The present invention provides an improved and rapid method for the preparation of plasmid nucleic acid in which iodine is used to eliminate residual traces of non-specific deoxyribonuclease and ribonuclease that frequently contaminate plasmid nucleic acid preparations made by conventional rapid techniques. The method also optimizes lysis conditions to prevent permanent denaturation of plasmid nucleic acid and improves conditions for selective precipitation of DNA by polyethyleneglycol in the presence of RNA. Plasmids prepared by this method are sufficiently stable that they may be stored in solution at room temperature for months or even years.
The novel method of the invention has advantages for the preparation of plasmid nucleic acid that are irrespective of any need to eliminate nucleases. If the host expresses Endo I or other similar nuclease it is eliminated through chemical modification of amino acid residues contained therein. In the present method iodine and, optionally cesium ions, cause the precipitate of cellular components other than plasmid to be more cohesive and more dense than those formed in other protocols and so this precipitate is more easily removed by centrifugation. Formation of the denatured closed circular form (form IV), which often contaminates plasmid samples prepared by other alkaline techniques is avoided.
Ribonuclease A, used in most protocols to destroy cellular RNA, is not a requirement of the present method, although slightly greater purity may be obtained if its use is included. When ribonuclease is used in the present method it is inactivated and therefore is unlikely to present a problem for subsequent use of the DNA in in vitro transcription experiments.
Sample handling, an important issue when comparing protocols because much time can be lost in changing pipette tips or in moving sample between containers while avoiding disturbance of loose pellets, is minimized. There is only one transfer of sample between centrifuge containers which can be accomplished by pouring. Reagent volumes have been adjusted to simple multiples of a unit volume to permit semi-automated pipetting with an inexpensive repeating pipettor. The procedure requires only a bench top centrifuge or filtration apparatus and no expensive reagents. Furthermore the method can be scaled up to either large volumes of culture or to large numbers of samples without difficulty. The method can be completed in less than 30 minutes with as many samples as can be conveniently centrifuged or filtered while working entirely at room temperature.
The resultant nucleic acid product is essentially free of RNA and is sufficiently pure that it can be used in molecular biological protocols including subcloning and DNA sequence analysis.
In accordance with an object of an aspect of the present invention is a method for preparing plasmid nucleic acid from a host cell, the method comprising: lysing host cells containing said plasmid nucleic acid; providing iodine in sufficient quantity to inactivate nucleases and precipitate host cell nucleic acid and protein, leaving a supernatant containing the plasmid nucleic acid; precipitating the plasmid nucleic acid from the supernatant; and isolating the plasmid nucleic acid.
In one embodiment of the method of the invention the solution of iodine together with a buffer containing a potassium or a cesium salt is added in sufficient quantity to permanently inactivate nucleases and to precipitate the host cell nucleic acid and protein, leaving behind a supernatant containing the plasmid nucleic acid. In an alternative, preferred embodiment, iodine may be generated in situ in the precipitating buffer through reaction of an iodide salt contained therein with an oxidizing agent such as an iodate salt.
In accordance with a further object of an aspect of the present invention is a commercial kit for the preparation of plasmid nucleic acid from a host cell, the kit comprising; mild alkaline lysis reagent for lysing host cells; an iodine providing solution for precipitating host cell nucleic acid and protein and for inactivating enzymes; and a solution for precipating the plasmid nucleic acid. The plasm
Gansheroff Lisa
Ketter James
Sim & McBurney
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