Method and device for purifying nucleic acids

Details Method and device for purifying nucleic acids Method and device for purifying nucleic acids

2001-04-05

2002-10-15

Yucel, Remy (Department: 1636)

Organic compounds -- part of the class 532-570 series

Organic compounds

Carbohydrates or derivatives

C536S025410, C435S270000

Reexamination Certificate

active

06465640

ABSTRACT:

The present invention concerns the separation of nucleic acids from whole cells in a sample. In particular, it concerns the recovery of genomic DNA from cells of a patient blood sample.
Current techniques of separating nucleic acids from a sample can be both time consuming and costly, involving separate steps of cell lysis, nuclei lysis, protein precipitation, DNA rehydration and RNA digestion. One of the many kits available for genomic DNA purifiction from e.g. whole blood samples is the Wizard (RTM) Genomic DNA Purification Kit supplied by Promega Kit sufficient to purify 50×3 ml of sample can be very expensive and performing the protocol takes about 45 minutes. Other techniques are available but typically take longer to perform and require the use of proteinases or organic solvents, or the use of columns or resins to purify fractions of a sample.
The present invention improves upon the prior art, providing a novel method for purifying nucleic acids from whole cells in a sample. According to the present invention there is provided a method for purifying nucleic acids from whole cells in a sample comprising passing the sample across the surface of at least one porous membrane contained in a filter device having a sample inlet and a sample outlet, the path from the inlet to the outlet being partially occluded by the membrane or membranes and generating a transmembrane pressure.
The technique of filtering flow across the surface of a membrane is widely known as “cross-flow filtration” (see for example Hood, R, 1998, New Frontiers in Screening for Microbial Biocatalysts, 77-86; WO 96/04067; WO 96/04068; WO 96/17673; WO96/20402). Other membrane devices include those of U.S. Pat. No. 5,438,128. However, none of them provide for flow around the membranes (as opposed to through it or across it by only partially occluding the path between sample inlet and outlet). The present inventor has found that, surprisingly, this can be used to rupture cellular and nuclear membranes to purify nucleic acids. By only partially occluding the path between the inlet and the outlet, i.e. by providing for flow around the membrane or membranes, it is possible for larger pieces of cellular debiris to pass around the membrane rather than clogging the pores. Not only are the cells ruptured, but the nucleic acids contained in the cell are deposited upon the surface and interior of the membrane. Experiments (below) have shown that a very substantial proportion of cellular nucleic acids are retained by the membrane. Furthermore, tests (see below) using molecular weight markers as standards for comparison have also shown that the average molecular weight of recovered DNA is very high, indicating that although the shearing forces exerted upon the cells are sufficient to rupture cellular and nuclear membranes, they are not so great as to cause extensive shearing of nucleic acids.
This is particularly important for subsequent testing of recovered nucleic acids since damaged strands may not display an appropriate binding site (e.g. epitope) or may not provide a sequence sufficient for correct PCR primer binding and elongation in order to effect amplification.
A minimum transmembrane pressure of 0.25 bar is required to effect cellular rupturing. As the transmembrane pressure is increased, rupturing is more efficient, there is a slight increase in shearing of nucleic acids and, at higher pressures, membrane deformation can occur. As the transmembrane pressure is increased it is also found that more of the nucleic acids are deposited in the pores/interstices of the filter membrane, and at high transmembrane pressures it is possible that nucleic acids, particularly shorter stretches of nucleic acids, may be forced through the membrane altogether and thus not be retained on the membrane. Due to all of this, a maximum transmembrane pressure of about 1.25 bar appears to be appropriate, although of course the maximum transmembrane pressure for a particular device may be readily determined using simple experiments.
Light-microscope examination of membranes has shown their surface to be covered in nucleic acids and no other cellular components to be visible.
Removal of other cellular components may be further improved when using membranes having a lumen (e.g. hollow fibre membranes) by allowing the exit of filtrate via the lumen. For example, one end of a hollow fibre membrane (or series of hollow fibre membranes) may be sealed and the other end of the membrane(s) connected to a lumen restrictor valve to allow escape of filtrate when it is at a sufficiently high pressure to open the restrictor valve. For example, a device having a 0.5 bar outlet valve restrictor may be used with a lumen outlet having a 0.25 bar restrictor valve. Alternatively, the pressure required to open the lumen restrictor valve may be greater than that for the outlet valve, in which case nucleic acids will be more predominantly deposited on the outside of the membrane rather than in the pores/interstices. Too low a pressure required to open the lumen restrictor may result in too high a transmembrane pressure, in turn resulting in increased nucleic acid shearing and loss of nucleic acids through the lumen outlet. Appropriate values for outlet and lument restrictor valves may be readily determined by one skilled in the art using simple experimentation.
Experiments (below) have also shown that successful PCR amplification may be performed directly on the membranes carrying bound nucleic acids—RNA may be amplified using an initial step of reverse transcription. PCR techniques are well known (PCR (Volume 1): A practical approach. Eds. M. J. McPherson, P. Quirke and G. R. Taylor. Oxford University Press, 1991) and may be readily used.
The simple methodology of the present invention means that the apparatus used may be relatively inexpensive. Importantly, nucleic acids, particularly genomic DNA, may be recovered from a sample such as a whole blood sample in a very short length of time—experiments (below) show that nucleic acids may be separated from 5×1 ml samples in a total of about 2 minutes.
Naturally, it is desirable to recover the greatest possible quantity of nucleic acids from a sample and so the membrane or membranes may occlude most of the path between the inlet and outlet. For example, the membrane may occupy at least 90% of the cross-sectional area of the filter device between the inlet and the outlet. In this way maximum cellular contact with the membrane (and thus cellular rupturing and nucleic acid recovery) is achieved whilst allowing flow of cellular debris around the membrane, thus ensuring that the final membrane-bound nucleic acids are substantially free of impurities. It may also be desirable to prefill the filter device with e.g. an appropriate buffer prior to introducing the sample so that pressure is exerted upon the whole of the sample as it passes from inlet to outlet. Similarly, a flush step may be used after the sample has been introduced into the filter device in order to ensure that it all (except for that retained by the membrane) passes through to the outlet.
The pores in the membrane act to allow the creation of a transmembrane pressure difference and allow the trapping of nucleic acids. The pores also allow smaller pieces of cellular debris to easily pass through to the outlet. Pores may have a molecular weight cut off (MWCO) of 10
5
-10
7
daltons, for example, about 10
6
daltons.
Examples of membranes which may be used are a polypropylene membrane with a pore diameter of 0.2 &mgr;m (for example supplied by AKZO NOBEL) and polysulphone membranes models ULF 1 million and ULF 750 supplied by A/G Technologies Corp. USA, as well as models 9002 and 9005 supplied by Fresenius A. G. (St. Wendel, Germany). The membranes should not repel the nucleic acids and so it may be necessary to pre-treat them to ensure they are hydrophilic and/or have a positive charge. In the case of polypropylene membranes, they may be pre-soaked in a solution of 20% Tween. Membranes having coarse surfaces may be used in order to enhance the ru

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