Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2002-08-14
2004-12-07
Horlick, Kenneth R. (Department: 1637)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
Reexamination Certificate
active
06828104
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates generally to matrices for conducting nucleic acid affinity chromatography. More specifically, the present invention relates to methods of preparing affinity chromatography matrices that bind a plurality of different preselected nucleic acids. The matrices, for example, can bind to substantially every known nucleic acid message in a sample.
BACKGROUND OF THE INVENTION
Affinity chromatography has become a valuable tool for separating biological materials from fluid (typically aqueous) media. Examples include biologically active molecules such as small ligands, proteins, nucleic acids, enzymes, etc.
The basic principle of affinity chromatography involves immobilization of a binding moiety (e.g., a ligand) to an insoluble support. The immobilized binding moiety can then be used to selectively adsorb, e.g., from a fluid medium, the target component(s) (e.g. an enzyme) with which the binding moiety specifically interacts thereby forming a binding moiety/target complex. Elution of the adsorbed component can then be achieved by any one of a number of procedures which result in disassociation of the complex. Thus the specific biologic properties of biological macromolecules can be exploited for purification. The process can be used to isolate specific substances such as enzymes, hormones, specific proteins, inhibitors, antigens, antibodies, etc. on the basis of the biologic specific interactions with immobilized ligands.
Nucleic acid affinity chromatography is based on the tendency of complementary, single-stranded nucleic acids to form a double-stranded or duplex structure through complementary base pairing. A nucleic acid (either DNA or RNA) can easily be attached to a solid substrate (matrix) where it acts as an immobilized ligand that interacts with and forms duplexes with complementary nucleic acids present in a solution contacted to the immobilized ligand. Unbound components can be washed away from the bound complex to either provide a solution lacking the target molecules bound to the affinity column, or to provide the isolated target molecules themselves. The nucleic acids captured in a hybrid duplex can be separated and released from the affinity matrix by denaturation either through heat, adjustment of salt concentration, or the use of a destabilizing agent such as formamide, Tween-20, or sodium dodecyl sulfate (SDS).
Hybridization (the formation of duplex structure) between two nucleic acid sequences is highly sequence dependent. Sequences have the greatest affinity with each other where, for every purine in one sequence (nucleic acid) there exists a corresponding pyrimidine in the other nucleic acid and vice versa. This sequence dependency confers exquisite specificity on hybridization reactions and permits the preparation of affinity columns that are highly selective for particular target nucleic acids.
Affinity columns (matrices) are typically used either to isolate a single nucleic acid typically by providing a single species of affinity ligand. Alternatively, affinity columns bearing a single affinity ligand (e.g. oligo dt columns) have been used to isolate a multiplicity of nucleic acids where the nucleic acids all share a common sequence (e.g. a polyA).
SUMMARY OF THE INVENTION
This invention provides pools (solutions) of nucleic acids, and nucleic acid affinity matrices that bear a large number of different nucleic acid affinity ligands allowing the simultaneous selection and blocking or removal of a large number of different preselected nucleic acids from a sample. This invention additionally provides methods and devices for the preparation of such affinity matrices.
In one embodiment, this invention provides a method of making a nucleic acid pool (solution of nucleic acids) comprising a plurality of different nucleic acids. The method includes first, providing a nucleic acid amplification template array comprising a surface to which are attached at least 20 oligonucleotides having different predetermined (known) nucleic acid sequences; and second, amplifying the multiplicity of oligonucleotides at least about 10 fold to provide the nucleic acid pool. The oligoncleotides, or subsequences thereof, preferably encode “capture probes” which can be incorported into an affinity matrix. In a preferred embodiment, each different oligonucleotide is localized in a predetermined region of the surface, the density of the oligonucleotides is preferably greater than about 60 different oligonucleotides per 1 cm
2
, and the different oligonucleotides preferably have an identical terminal 3′ nucleic acid subsequence and an identical terminal 5′ nucleic acid subsequence. The 3′ and 5′ nucleic acid subsequences can be the same as each other or can differ in length and/or nucleotide sequence. The 3′ and 5′ subsequences preferably flank “unique” central subsequences encoding the capture probes.
The method can further involve attaching the pool of nucleic acids to a solid support to form a nucleic acid affinity matrix.
The template nucleic acids comprising the amplification template can be synthesized entirely using light-directed polymer synthesis or channel methods. Alternatively the template nucleic acids can be synthesized using a combination of methods. For example, in one embodiment, the 3′ segments (subsequences) of the template nucleic acids can be synthesized using standard phosphotriester (e.g., phosphoramidite) chemistry. A middle (unique) portion of the template nucleic acids can then be synthesized using light-directed polymer synthesis or mechanically-directed synthesis methods. Finally, the 5′ segments (subsequences) of the template nucleic acids can be synthesized using phosphotriester chemistry.
The template nucleic acids can be amplified using any nucleic acid amplification method (e.g. polymerase chain reaction, ligase chain reaction, transcription amplification, etc.). In a preferred embodiment, amplification is by PCR. The template nucleic acids can be released into solution prior to the amplification (e.g. by cleavage of a linker joining the template nucleic acids to the substrate) thereby allowing the amplification to be performed in solution. Alternatively, and in a preferred embodiment, the amplification is performed without releasing the template nucleic acids from the substrate.
In a preferred embodiment, the amplification templates include primer binding regions (e.g. 3′ and 5′ subsequences flanking the region encoding the capture probe). Preferred amplification templates include identical 3′ and 5′ primers. The primer binding regions of the amplification template oligonucleotides, and hence the corresponding complementary PCR primers, preferably range in length from about 4 to about 30 nucleotides. The primer binding regions can be identical to each other or can differ in nucleotide sequence and/or in length.
In a particularly preferred embodiment, the region of the amplification templates encoding the capture probes (the non-identical portion of the amplification template(s)) ranges in length from about 6 to about 50 nucleotides. Where it is desired to remove the primer binding regions, they can include a recognition site of a nuclease to facilitate cleavage. In a particularly preferred embodiment, the thermal melting points of the template nucleic acid sequences encoding the capture probes with their complementary sequences varies by less than about 20° C.
In another embodiment, this invention provides for nucleic acid amplification template arrays for practice of the above-described method. In a preferred embodiment, the template arrays comprise a predetermined multiplicity of at least 20 oligonucleotides having different nucleic acid sequences. Each different oligonucleotide is preferably localized in a predetermined region of said surface. The density of the oligonucleotides is preferably greater than about 60 different oligonucleotides per 1 cm
2
, and the different oligonucleotides have identical terminal 3′ nucleic acid s
Chee Mark S.
Gingeras Thomas R.
Lipshutz Robert J.
Morris MacDonald S.
Affymetrix Inc.
Horlick Kenneth R.
McGarrigle Philip L.
Zhou Wei
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