Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2001-11-28
2004-11-02
Horlick, Kenneth R. (Department: 1637)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200, C435S173500, C435S173600, C435S173700
Reexamination Certificate
active
06811981
ABSTRACT:
The present invention relates to methods for producing release of intracellular material from cells.
Current methods for cell lysis and isolation of cellular material are laborious and time consuming and require a number of steps. For example, the preparation of DNA from bacteria requires a protocol of no fewer than ten individual steps. To produce effective cell lysis in gram negative bacteria, treatment with reagents such as EDTA and digestion with enzymes such as lysosyme and RNase are required. This is followed by cold shock, osmotic shock or boiling in order to release cellular material. Multiple steps are then necessary to harvest nucleic acids from the lysed preparation.
In the case genomic DNA isolation these are, following lysis of cells to release the DNA:
digestion of RNA and proteins with enzymes,
removal of contaminants, usually by solvent extraction,
and finally, dialysis or ethanol precipitation steps to give a clean preparation.
Plasmid extraction comprises cell lysis and selective precipitation of genomic DNA followed by the purification of plasmid DNA by gradient centrifugation or by ion-exchange chromatography. DNA extraction techniques currently in use include phenol-chloroform extraction and salting out methods for genomic DNA, and cesium chloride/ethidium bromide density gradients, and ion-exchange columns, for plasmid DNA. In addition kits are widely used for purification of DNA and DNA fragments which are based on the precipitation of DNA under chaotropic conditions. All of these techniques have their limitations. For example, density gradients and phenol/chloroform extraction are time consuming processes to use, taking up to 24 hours to perform. In addition, the copious use of phenol for these purposes is highly undesirable due to its toxic and caustic nature. Methods of isolating large fragments of DNA can result in DNA shearing.
It is known that electroporation using voltages in the kilovolt range can produce release of intracellular material through the permeablised cell membrane produced transiently in the electroporation process; see for instance Brodelius P. E. Funk C, Shillito R. D., Plant Cell Reports 7, 186 (1988) and Heery D. M., Powell R., Gannon, F., and Dunican L. K. Nuc. Acids Res. 17, 10131 (1989).
Electroporation involves the application of high voltages, typically in excess of 1 kV in pulses of short duration in the order of milliseconds. Generally, the field gradient between the electrodes across which the voltage is applied to a suspension containing the cells to be electro-porated will be in excess of 1 kV per cm. This requires sophisticated and expensive apparatus.
It has now been found that it is possible to obtain release of intracellular material from cells by the application of voltages of a lower order of magnitude not previously thought to be capable of affecting cell membrane structure in such a way.
Accordingly, the present invention provides a method of producing release of intracellular material from cells comprising applying a voltage of not more than 50 volts to a suspension of said cells.
Preferably, the voltage is from 0.5 to 50 volts with a strong preference for voltages in the lower part of this range e.g. from 0.5 to 15 volts, most preferably from 1 to 10 volts.
The voltage may be a DC voltage or an AC voltage.
Unlike the practice in electroporation, the voltage may be applied continuously, subject to avoiding excess heating effects which may become a problem if the voltage is in excess of 15 volts. Preferably the voltage is applied for a period of at least 30 seconds, more preferably for at least 2 minutes, e.g. from 2 to 20 minutes. Preferably the voltage is applied continuously for a period as specified above, but this process may be repeated, e.g. by the application of voltage for repeated periods of several seconds to several minutes, e.g. 5 seconds to 10 minutes.
The electrodes by which the voltage is applied may preferably be spaced by 10 mm or less, e.g. 5 to 7 mm. However, it may be preferred to optimise the conditions for producing denaturation of double-stranded DNA released from the cells, in which case a smaller electrode spacing will be desirable. To accomplish denaturation of released DNA, preferably the voltage is applied to the suspension between closely spaced electrodes, preferably not spaced by more than 5 mm at their closest approach, e.g. by no more than 1.5 mm and most preferably by no more than 0.5 mm.
One of the electrodes may be constituted by a container of conductive material in which the sample being treated is contained.
The process may be conducted to produce cell lysis and to produce the release of intracellular materials including proteins and nucleic acids, including double stranded DNA, and other biomolecules. A process for producing denaturation of double-stranded nucleic acid utilising apparatus suitable for use in the present invention is described in Application PCT/GB95/00542. This process is itself an improvement on processes for electrochemical denaturation of double-stranded nucleic acid described in WO92/04470 and WO93/15224. As disclosed in those specifications, nucleic acid may be denatured reversibly by the application of an electrical voltage and such denaturation may be used as a step in a number of more complex tasks including hybridisation studies and nucleic acid amplification procedures such as PCR.
Nucleic acids released from cells by methods according to the present invention may be further processed according to the teachings of these specifications.
Accordingly, the present invention includes a method of producing single-stranded nucleic acid which comprises releasing double stranded nucleic acid from cells by applying a voltage of not more than 50 volts to a suspension of said cells with an electrode to release nucleic acid from said cells and denaturing the double-stranded nucleic acid by applying the same or a different voltage to said suspension with said electrodes to convert said double-stranded nucleic acid to single-stranded nucleic acid.
The range of voltage within which production of denaturation in this way is achievable will not be as wide as the range of voltage appropriate for producing cell lysis and accordingly it is preferred that in the denaturation stage, a voltage of from 0.5 to 3 volts is applied, more preferably from 1.5 to 2.5 volts, measured as a voltage difference between the electrodes.
As described in WO92/04470, one may employ a promoter compound such as methyl viologen to produce more rapid denaturation.
More generally, the promoter may be any inorganic or organic molecule which increases the rate or extent of denaturation of the double helix. It should be soluble in the chosen reaction medium. It preferably does not affect or interfere with DNA or other materials such as enzymes or oligonucleotide probes which may be present in the solution. Alternatively, the promoter may be immobilised to the electrode or included in material from which the electrode is constructed. It may be a water soluble compound of the bipyridyl series, especially a viologen such as methyl viologen or a salt thereof. Whilst the mechanism of operation of such promoters is not presently known with certainty, it is believed that the positively charged viologen molecules interact between the negatively charged nucleic acids such as DNA and the negatively charged cathode to reduce electrostatic repulsion therebetween and hence to promote the approach of the DNA to the electrodes surface where the electrical field is at its strongest. Accordingly, we prefer to employ as promoters compounds having spaced positively charged centres, e.g. bipolar positively charged compounds. Preferably the spacing between the positively charged centres is similar to the spacing between the positively charged centres in viologen. Other suitable viologens include ethyl viologen. isopropyl viologen and benzyl viologen.
Other promoters are described in WO93/15224, i.e. multivalent cations such as magnesium. Other multivalent cations which are effective and which can be used include l
Bergmann Karin
Martin Sophie E. V.
Pollard-Knight Denise V.
Affymetrix Inc.
Horlick Kenneth R.
Morgan & Lewis & Bockius, LLP
Tung Joyce
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