Electrode capture of nucleic acid

Details Electrode capture of nucleic acid Electrode capture of nucleic acid

1999-03-11

2004-09-21

Horlick, Kenneth R. (Department: 1637)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C422S088000, C422S091000, C422S105000, C422S122000, C422S131000, C422S050000, C422S067000, C422S073000, C422S071000, C422S186000

Reexamination Certificate

active

06794130

ABSTRACT:

The present invention relates to processes for the manipulation of nucleic acids and in particular for capturing a nucleic acid or mixture of nucleic acids from cellular debris or other biomolecule mixtures.
When cells are lysed to release nucleic acids, the resulting mixture is complex. It may contain cell wall materials, proteins, polysaccharides and numerous other materials. To capture the nucleic acids contained therein has been a time consuming task which generally must be carried out before the nucleic acid can be used in other processes such as replication (or amplification) procedures or hybridisation assays.
Harrington et al in “DNA transformation with electrically charged tungsten microelectrodes”, International Worm Meeting, abstract 240 disclosed that a tungsten microelectrode subjected to a voltage of 3 V would attract DNA from solution to enable the DNA to be introduced into nematode worms. The DNA remained on the electrode when the latter was withdrawn from the solution. Reversal of the voltage is mentioned as a method of displacing the DNA from the electrode, although this is not disclosed to be advantageous. The DNA was present in pure form in a suitable buffer.
We have now discovered that a similar method can be employed to remove DNA or other nucleic acids from the complex mixtures formed during cell lysis, i.e. from admixture with cellular debris or form mixtures with other biomolecules generally, and that the nucleic acids so obtained can be removed from the electrode without damage so that they may be used in subsequent processes. Accordingly, the present invention now provides a method for capturing nucleic acid from a mixture of said nucleic acid with other biomolecules, e.g. cellular debris, comprising exposing an electrode to said mixture and applying to said electrode a nucleic acid attracting voltage, and removing said electrode from said mixture carrying said nucleic acid thereon.
The electrode may be removed from the mixture by physical movement of the electrode or by removal of the mixture, e.g. by washing.
A voltage of from 0 to 4, more preferably 0.5 to 3 volts is suitably applied to said electrode to attract said nucleic acid thereto. Generally, best results are obtained at approximately 1 V. Said electrode carrying said nucleic acid may then be exposed to a liquid into which said nucleic acid is to be introduced and said nucleic acid may be removed from said electrode into said liquid. This may be achieved by washing, preferably after reducing, turning off or reversing the electrical field. The voltage may be applied between a pair of electrodes which are both removed from the mixture, or only one electrode carrying said nucleic acid may be removed.
The removed nucleic acid may then be used as desired, e.g. subjected to a replication procedure or a hybridisation assay.
The mixture from which the nucleic acid is removed may be produced by a process of cell lysis as described in our PCT application PCT/GB95/02024. As described there, cells such as bacteria (e.g.
E. Coli
) may be lysed by subjecting them to a voltage of a few volts, e.g. 1 to 10 volts. Using the same electrode, the released nucleic acid may be captured and removed as described above. This provides a particularly elegant process for lysing cells and capturing nucleic acids from the cells.
Other crude mixtures from which to purify nucleic acids, especially DNA, include PCR or other amplification reaction mixes, sequencing reaction mixes, body fluid samples, e.g. blood or sputum or other DNA rich samples, e.g. micro-biological cultures.
For conducting processes such as nucleic acid amplification or hybridisation assays, it is generally necessary to denature DNA into single stranded form. As disclosed in WO92/04470, WO93/15224 and PCT/GB9500542, this also may be achieved by applying a voltage to an electrode. Such methods of denaturation may be used in the further treatment of DNA captured by the methods described herein.


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Teijeiro et al., Cyclic voltammetry of submicrogram quantities of supercoiled, linear and denatued DNAs with DNA-modified mercury electrode, J. of biomolecular structure and dynamics, vol. 11(2), p. 313-331, 1993.*
Harrington et al. DNA transformation using electrically charged tungsten microelectrodes, IEEE, p. 12-16, 1995.*
S. M. Lindsay et al., “Potentiostatic Deposition of DNA for Scanning Probe Microscopy,” Biophysical Journal, vol. 61, Jun. 1992, pp. 1570-1584.
E. Palacek, “Adsorptive Transfer Stripping Voltammetry Determination of Nanogram Quantities of DNA Immobilized at the Electrode Surface,” Analytical Biochemistry, vol. 170, No. 2, (1988) pp. 421-431.
Palecek, E., “New Trends in Electrochemical Analysis of Nucleic Acids,”Bioelectrochemistry and Bioenergetics20:179-194 (1988).

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