Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process...
Patent
1996-01-04
2000-10-24
Wortman, Donna
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
435259, 422 69, 422101, 2103216, 21032164, 21032172, C12P 100, B01D 6300
Patent
active
061365556
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to a method and apparatus for purifying target compounds such as nucleic acids from cells.
Conventional procedures for the purification of protein or nucleic acid, such as DNA, require lysis of the source cells followed by various fractionation steps involving centrifugation. Where DNA manipulation is to be carried out, small scale DNA preparations are required routinely, often in large quantities for the purpose of screening the DNA from the source cells. These processes are time consuming and labour intensive.
It has been proposed to avoid centrifugation in the extraction and purification of DNA by a relatively complicated series of steps. EP 0376080 discloses in general terms the use of precipitants to precipitate DNA from accompanying impurities. Ultrafiltration is then used as a means to isolate the DNA. However, no worked example of the proposed process is described and no indication of its feasability is indicated. Ultrafiltration forms the basis of other DNA purification methods as described in WO 87/07645 and EP 0517515.
Cation exchange resins have also been proposed as a means for separating relatively uncontaminated nucleic acids from impurities. EP 0281390 discloses the use of polycationic solid supports particularly to separate hybridized from unhybridized nucleic acids. EP 0366438 discloses the separation of nucleic acid from protein by binding the protein to a cation exchange resin.
A partially automated apparatus for carrying out biochemical reactions is disclosed in WO 92/02303 in which mountings for manipulating microtiter plates are proposed.
The present invention aims to provide an improved method for the purification from cells of target compounds such as nucleic acids, which method avoids the use of centrifugation, ultrafiltration or other complex procedures.
The target compound may comprise nucleic acid, protein or other desired compounds and is produced by the cell and expressed either internally or externally. Apart from nucleic acids, the method is particularly attractive for purifying recombinant proteins and antibodies.
In one aspect, the present invention provides a method for purifying nucleic acid from cells. The method comprises the following steps: debris; conditions to bind the nucleic acid to the matrix;
Because the method of the present invention is relatively simple, starting with the cell suspension it can be applied as a continuous method (i.e. without interruption from batch of cell suspension to desired product), for example in automated apparatus.
In a further aspect, the present invention provides a continuous method for purifying a target compound from cells. The method comprises the following steps: debris; bind the target compound to the matrix;
Preferably, the cell suspension is lysed in step (1) to form a cell lysate containing the target compound.
Preferably, the method further comprises the step of washing the matrix binding the target compound so as to remove contaminants before eluting the target compound from the matrix.
Any cell producing the target compound may be used in the method. For the purpose of this specification, the term "cell" is intended to encompass bacterial cells, cells from higher organisms, phage particles and other cell types or organelles which contain the target compound and may require some form of lysis step to release it. In the case of bacteria, nucleic acid may come from the bacterial nucleus or from cellular inclusions such as plasmids. Indeed, the method is especially useful for the preparation of plasmid DNA. Phage-infected bacteria may also be used in the preparation of phage DNA, such as Ml3 DNA. Cells from higher organisms include blood cells. Where genomic nucleic acid is to be prepared, nucleated blood cells form the cell suspension for lysis.
As a typical first step in the method, a cell suspension is formed by applying a cell culture to the filter so as to separate the cells from the culture medium. The liquid medium passes through the filter and is discarded. The cells on the filter are
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Stedman's Medical Dictionary, 24th Edition, Williams & Wilkins, Baltimore. 1982. p. 479.
Cambridge Molecular Technologies Limited
Wortman Donna
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