Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-07-17
2003-12-09
Whisenant, Ethan (Department: 1634)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091100, C435S091200, C436S094000, C536S023100, C536S025300, C536S025400
Reexamination Certificate
active
06660472
ABSTRACT:
The present invention relates to a method of lysing a microorganism which makes it possible, in general, to disrupt the latter, especially the membrane of one or more cells, in order to release at least one nucleic material of interest, for example deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) to be treated subsequently, in particular to be analysed.
The document U.S. Pat. No. 5,643,767 discloses a method of lysis for separating the DNAs or RNAs of cells in a liquid solution. A container is used for this purpose which contains a solvent for extracting the RNA or DNA, and a plurality of particles having a diameter of 0.1 to 1 mm, and at least one larger particle having a diameter of 3 to 5 mm. A movement is applied to the container thus filled, namely a shaking movement. It is expressly indicated in this document that this movement is preferred to a rotating movement, as generated with a mixer or another homogenizer, because with a rotating movement the cells simply turn in the same direction, and do not effectively collide with each other and with the beads so as to be crushed between the beads.
The document U.S. Pat. No. 4,295,613 discloses an apparatus for disrupting bacteria, according to which the sample containing the cells is placed in a tube in contact with Line beads. The beads, having a diameter of the order of 70 to 110 &mgr;m, are introduced into the tube, with the sample to be treated and any required buffer solution, and then an oscillating movement is applied to the container or tube along a horizontal axis, in order to obtain the subcellular constituents in solution such as enzymes, proteins, carbohydrates and the like.
The document FR-A-1,576,299 discloses a method for disrupting plant and animal cells, according to which a particulate material is used whose particle size is from 0.05 to 1 mm in diameter, it being possible for the particles to be made of steel or another similar material. A movement is applied to the mixture of biological sample and particles, according to a laminar flow.
The document EP-A-0,317,803 discloses a method for generating multilamellar liposomes encapsulating an aqueous medium. The method consists in mixing lipids and the aqueous medium to be encapsulated, in stirring the mixture in a container in the presence of particles having a diameter of less than 3 mm, the preferred size being 50 to 100 &mgr;m, in order to obtain liposomes having a diameter of about 150 to 3000 nanometers.
The document EP-A-0,796,917 discloses a device which releases particles into a biological sample containing cells. When the sample is stirred or sonicated, this device comprises a partition which retains the particles during a sufficient period, and then releases them and disrupts the cells, which thus makes it possible to make the nucleic acids accessible. The particles retained by the partition, which are made of glass, plastic or a metal such as zirconium, may have different shapes, and have a diameter of about 0.1 to 0.15 mm. One particle is assigned to breaking the partition, and has a diameter of about 1 to 4 mm, preferably 3 mm. The apparatus for subjecting to an alternating movement is a stirrer of the Biospec® trade mark.
The document EP-A-0,288,618 discloses a method of cell lysis, including microorganisms, in order to release subcellular constituents including DNA and RNA into solution. The biological sample is placed in a container with particles of different sizes. The container is then subjected to sonication until the cells release their constituents. The ultrasound causes the particles to vibrate through the sample, which causes the disruption of the cells by shearing. The particles are glass beads having a diameter of between 0.05 and 1 mm. The sonication time is 10 minutes at room temperature. The RNA and DNA are released in a manner accessible to genetic nucleic hybridization probes.
The document U.S. Pat. No. 5,464,773 discloses an apparatus for lysing cells, without destroying the subcellular constituents, in order in particular to release the RNA and DNA so as to subsequently carry out a hybridization. The container used contains two sizes of beads made of zirconium, glass or plastic and the like. In a preferred mode, the container contains 400 &mgr;l of particles of zirconium of two different sizes and weights, for example 0.7 g and 0.1 mm in diameter and 0.65 g and 0.5 mm in diameter. The movement applied to the container is a vibratory-type, in particular oscillatory-type, movement.
The document U.S. Pat. No. 4,666,850 discloses an apparatus and a method for lysing a blood sample during its centrifugation. The container receiving the blood sample contains particles or beads, made of plastic or glass, which should have a size which does not prevent the sedimentation of bacteria at the bottom of the tube during centrifugation.
The document EP-A-0,341,215 discloses a method for producing heterologous proteins from genetically transformed yeasts. To evaluate the quantity of proteins, it is simply specified that to lyse the cells, mechanical shearing forces are applied by shaking the biological sample containing the yeasts with glass beads.
The document EP-A-0,284,044 discloses a method for increasing the production of proteins in yeasts. It is indicated that the cells (yeasts), for analysing the quantity of proteins, are preferably lysed by applying a vortex-type movement to the biological sample, with glass beads having a diameter of 450-500 &mgr;m at a maximum speed for one minute, three times in succession.
The document U.S. Pat. No. 4,775,622 discloses a method for expressing and recovering proteins from a yeast culture.
The methods generally used for lysing a biological sample comprising at least one microorganism, specifically for releasing at least one nucleic material of interest, essentially consist according to the prior art in that:
a biological sample in a liquid medium, comprising the microorganism to be lysed, is placed in a container,
at least one particulate material which is relatively hard and substantially inert relative to the nucleic material is placed in said container,
and the mixture of biological sample and particulate material is essentially subjected to an alternating movement, of variable amplitude and/or frequency if it is a regular movement, depending on the technique or the equipment chosen for causing the movement.
These methods which are used have some disadvantages. They are not sufficiently effective, in particular the cell lysis proves to be inadequate in quantity and quality for releasing the nucleic material.
Furthermore, they do not always make it possible to lyse cells which are reputed to be resistant to lysis, in particular the cells of Gram+bacteria, for example those of Mycobacteria.
Likewise, the use of these methods often requires the addition of additional reagents such as, for example, enzymes and/or detergents.
According to the present invention, it is found that, contrary to the teaching disclosed in U.S. Pat. No. 5,643,767, a rotating movement of the vortex type was particularly suitable for lysing microorganisms and releasing the nucleic material of interest directly, provided that certain parameters for this movement are chosen, in relation in particular to the container.
The subject of the invention is therefore a method of complete, effective and simple lysing of a biological sample comprising at least one microorganism, with no reagent and/or additional operating step during the use of the method.
The first subject of the present invention is therefore a method of lysing a biological sample comprising at least one microorganism of the bacterium type, in order to release at least one nucleic material of interest belonging to said microorganism, according to which:
said biological sample in a liquid medium is placed in a container,
at least one particulate material which is relatively hard and substantially inert relative to the nucleic material is placed in said container,
the mixture of biological sample and particulate material is subjected to a movement,
characteriz
Clark Dickey Kathleen Ann
Colin Bruno
Jay Corinne
Lopez Sophie
Paris Cécile
Bio Merieux
Lu Frank W
Oliff & Berridge, PL.C
Whisenant Ethan
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