Use of nucleic acids bound to carrier macromolecules

Details Use of nucleic acids bound to carrier macromolecules Use of nucleic acids bound to carrier macromolecules

1999-05-18

2001-03-27

Brusca, John S. (Department: 1631)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S091200, C536S023100

Reexamination Certificate

active

06207385

ABSTRACT:

The present invention relates to processes involving the use of nucleic acids such as oligonucleotides bound to carrier macromolecules and to new forms of immobilised nucleic acids.
Many different processes have been devised involving the use of nucleic acids such as oligonucleotides. These include assay procedures such as hybridisation assays in which the ability of a probe oligonucleotide of a given base sequence to recognise and bind a nucleic acid of complementary sequence is utilised. They include also amplification procedures in which a nucleic acid template of a given sequence is replicated. Such amplification procedures involve the use of primers which are complementary in sequence to a portion of the sequence to be replicated.
The product of such an amplification procedure is generally a nucleic acid in solution in the reaction mixture. Work up procedures are generally then needed to isolate or to detect the amplification product. Attempts have been made to adapt such amplification procedures to operate with the or a primer oligonucleotide immobilised to a solid support, e.g. a magnetic bead, to make it easier to collect the amplification product. However, such steps have usually interfered with the amplification to some extent.
WO96/31622 discloses the binding of oligonucleotides directly to a solid support, e.g. aminated polypropylene. The tethered oligonucleotides are used as primers for DNA-dependent synthesis by DNA polymerase.
WO96/13609 discloses a solid phase nucleic acid amplification using an oligonucleotide primer immobilised on a functionalised solid support. The primer is linked to the support by a polyfunctional molecule. The linker molecules are of relatively low molecular weight, e.g. analogs of decamer oligonucleotides.
WO96/04404 discloses carbonylated latex bead having oligonucleotide primers directly bonded thereto for use in hybridisation and amplification reactions.
Alternatively, oligonucleotides may be bonded to the surface of an epoxysilane derivativized solid support via respective relatively low molecular weight linker molecules, e.g. hexamethylene glycol.
WO91/00868 discloses oligonucleotides linked to a solid support via a dithio (—S—S—) bridge. Part of the oligonucleotide acts as a spacer between a sequence having relevant specificity and the susbtrate. Target oligonucleotides are hybridised to the immobilised specific sequence, which is then extended to incorporate labelled nucleotides.
WO90/06042 discloses attaching DNA probes to magnetic particles. The magnetic particles are used in an assay in which the attached probe sequences are hybridised and extended to incorporate a label. The magnetic beads are coupled to streptavidin and the probe sequences are biotinylated for attachment to the streptavidin coupled beads.
WO 93/01498 describes methods for conjugating a carrier macromolecule to any of various molecular species, including oligonucleotides and polynucleotides, via a divinylsulphone based chemistry. The carrier macromolecule is typically a polysaccharide such as dextran. The carrier macromolecule may also be conjugated to a second molecular species which acts as a label.
We have now devised various processes in which such nucleic acids bound to carrier macromolecules may be used advantageously. In a first aspect of the invention, it has surprisingly been found that amplification processes proceed well using primer which is bound to a carrier macromolecule.
Accordingly, in a first aspect, the invention provides a process for the replication of a nucleic acid template comprising hybridising to said template a primer having a sequence complementary to a portion of said template, which primer is bound to a carrier macromolecule, and extending said primer to replicate said template in complementary form.
Preferably, the carrier macro molecule is a natural or synthetic polysaccharide, a homopolyamino acid, a natural or synthetic polypeptide or protein, or a synthetic polymer having nucleophilic functional groups, for instance a polyvinyl alcohol, a polyallyl alcohol or polyethylene glycol or a substituted polyacrylate.
More preferably however, the carrier macromolecule is a dextran, which term includes carboxymethyl-dextrans, a starch, an hydroxyethyl-starch, an hydroxypropyl-starch, a glycogen, an agarose derivative or cellulose derivative, including hydroxyethyl- and hydroxypropyl-celluloses, or a natural gum.
Preferably, the carrier macro molecule in its free state is substantially linear and substantially uncharged at a pH in the range of about 4 to about 10. Preferably, it is water soluble and it suitably has a peak molecular weight in the range of about 1,000 to about 40,000,000, e.g. over 10,000 or over 100,000 or over 1,000,000.
Typically, a multitude of primer molecules will be bound to each carrier macromolecule.
As described in WO93/01498, using one possible conjugation chemistry the primer is bound to said carrier macro molecule via one or more moieties derived from divinyl sulphone, each of which moieties is attached to each of the carrier macromolecule and the primer by a covalent linkage formed between one of the two vinyl groups of a divinyl sulphone molecule and a reactive functionality on the carrier macromolecule or primer.
In the replication or amplification process, the primer may be extended by the action of a polymerase incorporating nucleotides on to said primer, e.g. in a polymerase chain reaction (pcr), strand displacement amplification (sda), self-sustained sequence replication (3sr) or nucleic acid sequence-based amplification (nasba) amplification procedure. Such procedures as previously practised are all well described in the literature.
Accordingly, according to a preferred practice of the invention, said template is a double stranded template and is denatured to single stranded form, said carrier macromolecule-bound primer is complementary in sequence to a region of a first one of the template strands and a second primer is provided which is complementary in sequence to a region of the other strand, which second primer is also extended so as to form a complementary sequence copy of said template second strand.
Alternatively, the primer may be extended by the action of a ligase ligating said primer to at least one further primer hybridised to said template, e.g. in an LCR (ligase chain reaction).
Where more than one primer is required in the replication or amplification procedure, one or more of said primers may be of the kind characterising this invention and the remainder may be conventional or otherwise modified oligonucleotides. Accordingly, the invention includes processes in which a second primer is extended or ligated in said amplification procedure which is also bound to a carrier macromolecule.
Optionally, during the extension of a said primer, a detectable marker is incorporated into the extended primer.
In a particularly advantageous aspect of the invention, the carrier macro molecule is itself bound to a solid support. Amplification products produced in the replication process will therefore themselves become bound to the support and can be removed from the reaction mixture for further treatment simply by removal of the support. The solid support may take many forms such as plates, strips, containers (including microtitre plate wells or eppendorf tubes), beads, membranes, or magnetic beads. This differs significantly from prior art schemes in which individual oligonucleotide molecules are linked to a solid support by respective low molecular weight linker molecules.
The extension of the primer may be conducted in situ in a biological sample. Thus the process may be one in which said biological sample is a plant or animal tissue sample, microorganism culture, or microorganism culture medium and the process may be in situ PCR where the much lower diffusion of the PCR product on the carrier macromolecule is advantageous in localising the amplified DNA in situ.
The product of the replication methods described above will normally be a nucleic acid bound to the carrier macro-molecule formed by exte

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