Solid phase technique for selectively isolating nucleic acids

Details Solid phase technique for selectively isolating nucleic acids Solid phase technique for selectively isolating nucleic acids

1999-05-13

2003-03-18

Jones, W. Gary (Department: 1634)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S007500, C435S007940, C536S025400

Reexamination Certificate

active

06534262

ABSTRACT:

BACKGROUND OF THE INVENTION
Many molecular biology applications, such as capillary electrophoresis, nucleotide sequencing, reverse transcription cloning and gene therapy protocols, which contemplate the transfection, transduction or microinjection of mammalian cells, require the isolation of high quality nucleic acid preparations. Quality is a particularly important factor for capillary electrophoresis for all sequencing methods and for gene therapy protocols. Quantity is an equally important consideration for some applications, for example, large scale genomic mapping and sequencing projects, which require the generation of hundreds of thousands of high quality DNA templates.
Extension product quality is crucial to the success of automated dye-labeled dideoxynucleotide sequencing methods, such as those described in Maniatis, T., et al.,
Molecular Cloning: A Laboratory Manual
, 2nd Edition, Cold Spring Harbor Laboratories, Cold Spring Harbor, N.Y., Sanger, E., et al.,
Proc. Natl. Acad Sci
. 74:5463-5467 (1977), and Mierendorf, R. and Pfeffer, D. Methods Enzymol. 152:5556-562 (1987), and is a particularly critical consideration for capillary electrophoresis protocols. The isolation of high quality nucleic acid preparations from starting mixtures of diverse composition and complexity is a fundamental technique in molecular biology.
The advent of demanding molecular biology applications has increased the need for high-throughput, and preferably readily automatable, purification protocols capable of producing high quality nucleic acid preparations. Although recent technological advancements and the advent of robotics have facilitated the automation of sequencing reactions and gel reading steps, throughput is still limited by the availability of readily automatable methods of nucleic acid purification.
SUMMARY OF THE INVENTION
The present invention is a method of separating different species of nucleic acid molecules present in a mixture, which can be a solution or a suspension, on the basis of differences in their molecular size. Separation is accomplished by selectively adsorbing target nucleic acid molecules (e.g., targeted or selected for isolation or purification), present in a mixture to a magnetically responsive solid phase carrier such as paramagnetic microparticles. In addition to target nucleic acid molecules the mixture can comprise other components, which include, but are not limited to, other non-target nucleic acid molecules, proteins, cell components, and reagents or chemicals used in methods in which the nucleic acids are processed or used. That is, the mixture can comprise a wide variety of types of molecules, from which target nucleic acid molecules are separated, by the present method on the basis of differences in molecular size. Thus, the invention discloses a method useful for isolating a particular species of nucleic acid molecule from a mixture. The nucleic acid to be isolated (e.g., separated or purified) (referred to as target nucleic acids or target nucleic acid molecules) are separated from the rest of the mixture based on molecular size and through the physical removal of the solid phase carrier to which the target nucleic acid molecules have been adsorbed according to the method described herein.
The present invention provides a method of selectively isolating (e.g., purifying) a species of nucleic acid molecule, based on its molecular size, from a mixture. This embodiment of the invention involves selectively precipitating and facilitating the adsorption of a target species of nucleic acid molecule to the functional group coated surface of a solid phase carrier. Purification of the target nucleic acids is accomplished by applying an external force which results in the removal (e.g., separation) of the solid phase carrier from the mixture in which the carriers have been suspended. In a preferred embodiment, the solid phase carrier is a paramagnetic microparticle and separation is accomplished by magnetic means.
The present invention is useful to isolate, from a mixture from which at least one species of nucleic acid molecule has been selectively removed, one or more additional (e.g., a second, third, fourth etc.) species of nucleic acid molecules which are of a smaller molecular size than the one or more target nucleic acid species which have already been removed from an initial (or starting) mixture by the method described herein. The additional species of nucleic acid molecule targeted for isolation in this additional embodiment remained soluble in the presence of the PEG and salt concentrations used to isolate the larger nucleic acid molecule and, therefore, will still be present in the mixture from which the first target nucleic acid molecule has been removed.
In an alternative embodiment of the instant invention, two or more species of nucleic acid molecule present in the same mixture, which differ in molecular size from each other by at least a factor of two, are separated from each other. The method described herein is used to isolate a particular species (e.g., a target species) of nucleic acid molecules of virtually any size, present in a wide variety of sources, from other nucleic acid molecules which are also present in the mixture. For example, the method disclosed herein can be used to isolate recombinant nucleic acid species, produced in host cells, including selective RNA precipitations based on molecular size, or replicative form DNA produced by a virus during lytic replication from endogenous host cell nucleic acid species. The method can also be used to isolate a particular species of nucleic acid from a solution resulting from a restriction enzyme digestion or an agarose solution containing nucleic acid. Alternatively, the method disclosed herein provides a size selection purification scheme suitable for use after a DNA shearing process (e.g., hydroshearing or sonication), thereby providing an alternative to the more traditional method of gel electrophoresis and band excision which are conventionally used to isolate a species of nucleic acid molecule targeted for purification. The disclosed method also finds utility as a method of separating multiplex PCR products, or as a sequencing reaction detemplating protocol. For example, using the method disclosed herein solid phase magnetically responsive paramagnetic microparticles can be used to selectively remove sequencing products and DNA templates from sequencing samples.
The present invention is also useful to isolate, from a mixture from which at least one species of nucleic acid molecule has been selectively removed, one or more additional (e.g., a second, third, fourth etc.) species of nucleic acid molecules which are of a smaller molecular size than the one or more target nucleic acid species which have already been removed from an initial (or starting) mixture by the method described herein. The additional species of nucleic acid molecule targeted for isolation in this additional embodiment remained soluble in the presence of the PEG and salt concentrations used to isolate the larger nucleic acid molecule and, therefore, will still be present in the mixture from which the first target nucleic acid molecule has been removed.
One embodiment of the instant invention provides a method of selectively isolating a target species of nucleic acid molecule from a host cell lysate which is a mixture of target nucleic acid molecules, non-target nucleic acid molecules and other cellular components. The presence of these other components (e.g., cellular components, reagents or biomolecules) could have an adverse effect on the downstream molecular biology application for which the target nucleic acids are being prepared. For example, the present method is useful to selectively isolate exogenous nucleic acid from endogenous host cell nucleic acid molecules. More specifically, the present method is useful to selectively isolate recombinant DNA, produced by the replication of exogenous DNA in an appropriate host cell, from endogenous host cell DNA and from other host cell components. Exogenous D

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