Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Patent
1996-11-14
1997-09-09
Housel, James C.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
435270, 436526, 209214, 210695, 210222, C12Q 168
Patent
active
056655541
DESCRIPTION:
BRIEF SUMMARY
WO91/12079 describes a method of recovering a biopolymer from solution which involves the use of magnetically attractable beads which do not specifically bind the polymer. The beads are suspended in the solution. Then the polymer is precipitated out of solution and becomes non-specifically associated with the beads. When the beads are magnetically drawn down, the polymer is drawn down with them. The polymer can subsequently be resolubilised and separated from the beads.
The present invention is a development of that technique. It is based on the surprising observation that the act of drawing down the magnetically attractable beads, which does bring down the precipitated polymer non-specifically associated with the beads, does not bring down an aggregated precipitate of a polymer which was present in the starting liquid prior to addition of the beads. In other words, it is the act of precipitating a polymer out of solution in the presence of the magnetically attractable beads that causes the polymer to entrap and become associated with the beads. This discovery is potentially useful in several circumstances, of which two are discussed here: found widespread usage in many areas within Molecular Biology [1].
In the following, the term "plasmid" is taken to include all double stranded DNA species that exist as supercoiled entities within bacterial host cells. Cosmids [2] are just one of many examples which will be obvious to those skilled in the art.
Current methods for plasmid preparation rely upon the removal (or destruction) of all of the undesired material present within a lysate of the plasmid-containing bacterial cells. Two main methods are in common usage. The first method, often referred to as "alkaline lysis" [3], relies upon the precipitation of a detergent complex of many of the undesired materials by high salt. This precipitation is followed by alcohol precipitation of the plasmid DNA. The second method, often referred to as "the boiling method" [4], relies upon the physical removal of viscous bacterial chromosomal and cellular material with a toothpick (or similar implement) followed by alcohol precipitation of the plasmid DNA.
Both of the above methods suffer from being difficult to automate and the "alkaline lysis" method also suffers from the inconvenience of having to transfer the supernatant to a fresh tube after the high salt precipitation step. sulphate, with some proteins requiring smaller concentrations of ammonium sulphate for precipitation. Thus for example, 30% ammonium sulphate can be added to precipitate some proteins present in a starting solution; later the ammonium sulphate concentration can be raised to 50% in order to precipitate the other proteins. As a technique for recovering these other proteins, this has the disadvantage that the supernatant needs to be transferred to a fresh tube after the first precipitation step.
The present invention addresses these problems. It has the advantage that the second precipitate can be recovered without the need to transfer the supernatant to a fresh tube after the first precipitation step. The invention is quick to perform and lends itself ideally to automation.
The invention provides a method of recovering a first molecular species from a starting liquid containing in solution the first molecular species in admixture with a second molecular species, which method comprises the steps of: precipitate of the second molecular species in the liquid, the liquid, of the first molecular species which entraps the magnetically attractable beads, and the associated aggregated precipitate of the first molecular species, the second molecular species.
So far as the nature of the first and second molecular species are concerned, the invention is of general application. The first and second molecular species may be proteins with different salting out points. Preferably the first and second molecular species comprise plasmid DNA and chromosomal DNA.
First, a precipitation reagent is added so as to cause the second molecular species to form an aggregated pre
REFERENCES:
Alderton et al., "Magnetic Bead Purification of M13 DNA Sequencing Templates," Analytical Biochemistry, vol. 201, pp. 166-169 (1992).
Richard K. Wilson, "High-Throughput Purification of M13 Templates for DNA Sequencing," BioTechniques, vol. 15, No. 3, pp. 414, 416, 418, 420, and 422 (1993).
Reeve Michael Alan
Robinson Philip Steven
Amersham International plc
Housel James C.
Wolski Susan C.
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