Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
2000-11-09
2004-11-23
Horlick, Kenneth R. (Department: 1637)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
Reexamination Certificate
active
06821758
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates to a method for the production of polynucleotide molecules with modified properties as well as to a kit containing instructions for carrying out said method.
Biomolecules—and, in particular, biopolymers such as polynucleotides, polypeptides, polysaccharides etc.—are not only the basis of biological life known to us but they are also used more and more in the most vaned technical fields of application. The search for new functional biomolecules, their isolation or production as well as their technical application is the subject-matter of modern biotechnology. Apart from incidentally finding so far unknown biomolecules in nature exhibiting desired properties (cf. natural substance screening), methods have emerged recently which imitate the principles of natural evolution in the laboratory and thus generate completely new biomolecules with specific properties (WO 92/18645; Eigen and Rigler, Proc. Natl. Acad. Sci. USA 91 (1994), 5740; Koltermann and Kettling, Biophys. Chem. 66 (1997), 159; Kettling et al., Current Topics in Microbiol. & Immunol. 243 (1999), 173). This so-called evolutionary biotechnology or directed molecular evolution takes the findings from theoretical and practical evolution research carried out over many years and applies them to the directed evolution of biomolecules.
Put very simply, directed evolution of molecular functions takes place by effective interaction of variation and selection processes acting on molecule populations. While variation starts out from the information content of a biomolecule, selection takes place by means of the molecular phenotype. Information of a polynucleotide molecule (genotype) denotes the sequential order of various monomers in a polynucleotide molecule. The phenotype of a polynucleotide molecule denotes the sum of the functions and properties of a polynucleotide molecule and of the transcription or translation products encoded by a polynucleotide. The linkage of sequence information and selectable phenotype can be achieved either by amplification linked selection (Kettling, PhD thesis, Göttingen/TU Braunschweig (1999)), by compartmentation and functional analysis, called screening (WO 92/18645; WO 99/34195) or by physical linkage of genotype and phenotype as well as their selection (DE 196 46 372; U.S. Pat. No. 5,849,545; DE-A1 43 056 51).
The kind of interaction of variation and selection processes are crucial for the success of directed evolution strategies. In nature as well as in the laboratory the quasi-species principle has proven to be the most successful strategy—measured by the time needed for an evolutionary generation and optimization of molecular functions. Quasi-species denotes a dynamic population of related molecule variants (mutants) resulting from erroneous replication. It could be shown that—corresponding to the quasi-species principle—not the wild type (centre of the quasi-species) but the whole spread is object of selection. Under modified selection conditions advantageous variants are already present in such a mutant distribution corresponding to their fitness value and do not have to be formed by subsequent, random mutations. If the selection parameters are changed the evolutionary generation resembles an implicitly directed drift of the quasi-species along the edges of the fitness landscape. The production of quasi-species and the application of this principle for evolutionary biotechnology is described in WO 92/18645.
The basis for the production of a quasi-species is an erroneous replication of the molecule variants. When polynucleotides are used replication preferably takes place by means of replication enzymes, i.e. polymerases which make the template-directed synthesis of a polynucleotide molecule possible. The introduction of errors, i.e. the variation of the molecule information, can be achieved by the inherent erroneous copying process alone, but also by the purposeful increase of the inaccuracy of the polymerase (e.g. defined non-balanced addition of the monomers, addition of base analogues, erroneous PCR, polymerases with very high error rate), by chemical modification of polynucleotides after synthesis, by the complete synthesis of polynucleotides under at least partial application of monomer mixtures and/or of nucleotide analogues as well as by a combination of these methods.
Apart from these methods to create punctual mutations (in the form of base exchanges, deletion and insertion) the recombination of sequence parts in nature is a very successful strategy for combining punctual mutations but also for combining domains within a polymer, for combining subunits of a heteromultimer or for combining gene variants within a gene cluster or a genome. Homologous recombination, in particular, i.e. the combination of corresponding sequence parts from different variants while maintaining orientation and reading frame plays an important role since the background noise of unrelated sequences that accompanies an unspecific recombination can be prevented. According to the quasi-species principle, homologous recombination is a purposeful means to expand the sequence distribution. Various related sub-distributions of a quasi-species which originate from the underlying fitness landscape but which have such a low relative degree of relatedness that converging along the edges of the fitness landscape is very unlikely without recombination, can be expanded tremendously by homologous recombination. Thereby, an evolutionary method emerges which, in contrast to serial introduction of mutations, leads to a multiplication of the experimental speed. Furthermore, a technologically controlled application of homologous recombination, in principle, also allows for the fusion of quasi-species distributions which were generated under different selection pressure and, thus, for the fusion of separately selected molecular functions.
In experiments, recombination can be conducted in different ways: on the one hand in vitro using individual enzyme functions or defined mixtures or sequences of enzymatic processing steps, on the other hand, in vivo using cellular recombination and/or repair processes.
For in vitro methods, mainly PCR based methods have technically been used so far. First to mention is DNA shuffling, also called sexual PCR (WO 95/22625: Stemmer, Nature 370 (1994), 389). In this method any overlapping gene fragments are provided and subsequently assembled into products of original length by a PCR without addition of a primer. Thus, the mutual priming of the fragments in each PCR cycle allows for fragments of different origin to be incidentally linked to form a product molecule in a homologous way. By adjusting the fragment length DNA shuffling makes it possible, at least in principle, to limit the frequency of recombination events. Another PCR-based method is the method of PCR using random primers (WO 98/42728); Shao et al., Nucl. Acids Res. 26 (1998), 681). In this method primers with randomized sequences are used which enable a start of polymerization at random positions within a polynucleotide. Thus, similar to DNA shuffling, short polynucleotide fragments are formed which can recombine with each other by mutual priming. With this method controlling of the recombination frequency is hardly possible. Moreover, unspecific primers lead to a comparatively high inherent error rate which can constitute a problem with sensitive sequence parts and/or long genes. Alternatively to these methods, the staggered extension process (WO 98/42728; Zhao et al., Nat. Biotechnol. 16 (1998), 258) uses a modified PCR protocol to provoke a strand exchange to take place during the PCR amplification. Using very short phases at the polymerization temperature between the melting and annealing phase allows for incompletely formed products to hybridize to new templates and to be prolonged further. Adjustment of the recombination frequency can take place by setting the polymerization time and the number of cycles. A technical limit, however, is the exact adjustment of very short
Eigen Manfred
Kettling Ulrich
Koltermann Andre
Birch & Stewart Kolasch & Birch, LLP
Horlick Kenneth R.
Max-Planck-Gesellschaft zur Forderung der Wissenschaften e.V.
LandOfFree
Method for the production of biopolymers with modified... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Method for the production of biopolymers with modified..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Method for the production of biopolymers with modified... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3346583