Labelling of polymers and sequencing of nucleic acids

Details Labelling of polymers and sequencing of nucleic acids Labelling of polymers and sequencing of nucleic acids

2000-11-27

2002-05-07

Riley, Jezia (Department: 1656)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S091100, C435S091200, C536S022100, C536S023100

Reexamination Certificate

active

06383750

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to polymers having functional groups attached thereto at random positions along their polymeric chains, hereinafter referred to as “labelled polymers”. It also relates to processes for preparing labelled polymers.
BACKGROUND OF THE INVENTION
The introduction of labels onto polymer chains is a subject of considerable interest. With the introduction of a small number of groups on a polymer chain, it is possible to make dramatic changes to some polymer properties while leaving others essentially unchanged. For instance, the introduction of several fluorophores per polymer chain will render the polymer easily detectable by highly sensitive fluorescence techniques while having little effect on properties such as mechanical strength, solubility, glass transition and melting temperature, etc. Similarly, the introduction of several biologically active groups per chain may turn an inert polymer into one which is biologically active, is readily bound to biomaterials (biocompatible), or one with medical applications (i.e. drug delivery, diagnostics).
There are two general means by which “labelled” polymers are prepared.
In the first method for labelling, a small amount of monomer bearing the label is polymerized with the bulk monomer(s). If the labelled monomer is incorporated in a small amount the desired polymer is obtained. Although this approach has been widely and successfully utilized, it has several drawbacks. Monomer synthesis is often required, some labels may not tolerate polymerization conditions, and the labelled polymer might not match the properties, in particular molecular weight, of an unlabelled control sample.
The second technique for “labelled” polymer synthesis is by polymer modification, in which labels are bound to an existing polymer. This is relatively straightforward for polymers with pendant functional groups such as amino, hydroxy or carboxylic acid groups. Thus, it is possible to use conventional chemistry to label polymers such as poly(acrylic acid), poly(allylamine) or poly(vinyl alcohol). In the case of biopolymers (peptides, membranes, polysaccharides, etc.), there are well developed techniques for labelling. However, it is more difficult to label polymers such as poly(ethylene), poly(propylene), poly(styrene), poly(ethylene oxide), poly(vinyl chloride), polyvinyl acetate), poly(methyl methacrylate), etc. which lack reactive functional groups. Some of these polymers such as poly(styrene) and poly(methyl methacrylate) can be labelled using more drastic conditions such as Friedel-Crafts reactions or trans-esterification, but many are resistant to even these more forceful approaches.
BRIEF REFERENCE TO THE PRIOR ART
B.-Z. Tang, S. Holdcroft, J. E. Guillet,
Macromolecules,
27, 5487 (1994); and S. Holdcroft, J. E. Guillet,
Macromolecules,
24, 1210 (1991); S. Holdcroft, B.-Z. Tang, J. E. Guillet,
Chem. Commun.,
1991, 280 disclose processes for binding fluorophores to polymers which cannot easily be labelled by conventional means. In these processes, photochemically generated free radicals are used to abstract a hydrogen atom from the polymer, in the presence of a second, fluorophore-bearing radical generated in the same or a parallel reaction. The fluorophore-bearing radical can then react with the polymer-centered radical to produce a fluorophore labelled polymer. A problem with this approach is that radical recombination and other side reactions are likely to take place, to consume many of the fluorophore-bearing free radicals, and if the lifetime and concentration of such free radicals is low, many polymer centered radicals will not be captured by them.
It is an object of the present invention to provide a novel process for preparing labelled polymers.
It is a further object of the invention to provide novel labelled polymers.
It is a further and more specific object of the present invention to provide novel, labelled nucleic acids, a process for their preparation, and a process of determining the sequence of a target nucleic acid using such novel, labelled nucleic acids.
SUMMARY OF THE INVENTION
The present invention provides, from one aspect, a process of labelling polymers by a free-radical mechanism, in which one reagent or mechanism is used to generate a free radical on the polymer (the polymer-centered radical), and a second reagent is used to generate a trapping radical which captures the polymer-centered radical. The labelling group (e.g. a fluorophore) may be bound to the trapping radical, i.e. it may be part of the second reagent. Alternatively, it can be added in a separate reaction, to bind to the trapping radical after attachment thereof to the polymer.
The second reagent used as a trapping radical is suitably one which will generate a stable free-radical. Stable free radicals are species which will not react with themselves, and which can exist for extended periods of time, at least one second, in solution or in the solid state. They will react rapidly with the polymer-centered (C-centered) free radicals, but not with most oxygen centered radicals. As a result, it is possible to prepare solutions of stable free-radicals at relatively high concentrations, to ensure efficient capture of the polymer-centered radicals.
Thus according to this aspect of the invention, there is provided a process of labelling a polymer with functional groups randomly distributed along the polymer backbone chain, which comprises:
generating polymer-centered free radicals at random position along the chain of a polymer;
reacting the polymer-centered free radicals so formed with stable free radicals to attach stable free radical derived groups thereto at random location along the polymer chain;
and attaching functional, labelling groups to the stable free radical derived groups, before or after the reaction thereof with the polymer-centered free radicals on the polymer chain.


REFERENCES:
patent: 0131830 (1985-01-01), None
patent: 91 13080 (1991-09-01), None
patent: WO 97/05156 (1997-02-01), None
Holdcroft et al. “Two-Photon Chemistry. 1. Fluorescence labeling of polystryene and poly(methyl methacrylate) by laser photolysis of 2-naphthylmethyl 1-naphthylacetate” Macromolecules, pp. 1210-1212, 1991.*
Tang et al. “A preparative Synthesis of lumiphore-labeled polymers” Macromolecules, pp. 5487-5490, 1994.

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