Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
2000-04-27
2002-02-26
Whisenant, Ethan (Department: 1655)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S006120, C435S089000, C436S094000, C536S025300
Reexamination Certificate
active
06350595
ABSTRACT:
DESCRIPTION
The subject of the present invention is a process for the synthesis of polynucleotides enabling to introduce random sequences along more or less extended tracts of the molecule, in such a way that randomness refers to units of three adjacent nucleotides, and that each one of the said units is fit in so as to match a limited number of codons, predefined in number and sequence, and in order to eliminate the effects of the genetic code degeneracy.
The applicative potentialities of a polynucleotide synthesis process with the above features are undoubtedly remarkable. Indeed, in recent years applications requiring its use have taken on an ever-increasing importance in many fields of the scientific research. It is the case, for instance, of site-specific mutagenesis, operated on a gene coding for a known protein in presumedly key positions in order to verify their actual role in the molecule structure or function. Another example is provided by libraries, containing “boxes” of random sequence synthetic oligonucleotides, that are realized in order to select molecules capable to carry out new biological functions.
In all these cases it is of the utmost importance that the randomness of the sequence is somehow controlled, so that only the desires codons shall be inserted, besides eliminating the effects of the genetic code degeneracy. Of equal importance is, obviously, the fact that said polynucleotide synthesis is carried out with a simple, cost-effective and efficient process.
TERMINOLOGY
It is useful to specify the terms hereinafter:
Support=the term support refers to a solid phase material to which monomers are bound in order to realize a chemical synthesis; said support is usually composed of resin or porous glass grains, but can also be made of any other material known to the man skilled in the art. The term is meant to comprise one or more monomers coupled to the support for the additional reactions of polynucleotides synthesis.
Coniugate or condense: these terms refer to the chemical reactions carried out in order to bind a monomer to a second monomer or to a solid support. These reactions are known to the man skilled in the art and are usually realized in an automated DNA synthetizer, following the instructions provided by the maker.
Monomers or mononucleotides: the terms monomer or mononucleotide refer to individual nucleotides utilized in the chemical synthesis of oligonucleotides. Monomers that can be utilized comprise both ribo- and deoxyribo-forms of each of the five standard nucleotides (derived from the bases adenine (respectively A or dA), guanine (G or dG), cytosine (C or dC), thymine (T) and uranic (U)). Base derivatives or precursors like inosine are also comprised in monomers, as well as chemically modified nucleotides, such as those for instance with a reversible blocking group in any position on the purinic or pyrimidinic bases, on ribose or deoxyribose or on hydroxylic or phosphate groups of the monomer. Those blocking groups comprise e.g. dimethoxytrityl, benzoyl, isobutyryl, beta-cyanoethyl and diisopropylamin groups, and are used to protect hydroxylic groups, phosphates and hexocyclic amines. However, other blocking agents known to the man skilled in the art may be adopted.
Dimers or dinucleotides: the terms dimers or dinucleotides refer to molecular units derived from the condensation of two monomers or mononucleotides as aforespecified.
Synthesis monomeric units: this term indicates unite utilized as essential elements in the synthesis process. In the process subject of the present invention they can consist of monomers or dimers; they can also be constituted of trinucleotide units in other processes known in art.
Codon or triplet: the term codon or triplet refers to a sequence of three adjacent desoxyribonucleotide monomers that specify one of the 20 natural amino acids utilized in a polypeptide biosynthesis. The term comprises also nonsense codons, codons that do not encode any amino acid.
Codon or randomized triplet: these terms refer to the case where the same sequence position corresponds to more than one codon in a polynucleotides set. The number of different codons can vary from 2 to 64 for each specific position.
Anticodon: the term anticodon refers to a sequence of three adjacent ribonucleotidic monomers that specify for a corresponding codon according to the known rule of purinic and pyrimidinic bases coupling.
Polynucleotides or Randomized oligonucleotides: this term refers to a set of oligonucleotides having randomized codons at one or more positions. For example, if the randomized oligonucleotides consist of six nucleotides in length (i.e. two codons), and both the first and the second position of the sequence are randomized so as to code for all of the twenty amino acids, then the population of randomized oligonucleotides shall comprise an oligonucleotide set with every possible combination of the twenty triplets in the first and second position. In this case, therefore, the number of possible codon combinations is 400. Analogously, if 15 nucleotide-long randomized oligonucleotides are synthetized in such a way as to be randomized in every position, then all the triplets coding for each of the twenty amino acids will be found in every position. On this case, the randomized oligonucleotides population shall contain 20
5
different possible oligonucleotide species.
When not clearly defined, other terms in use in, the present description are meant to be known to men skilled in the field, to whom the invention is aimed at.
For some terms pertaining molecular biology techniques, cfr. the Sambrook et al. manual (Sambrook et al, 1989). Other terms referred to substances of chemical nature not clearly defined are meant to be known to men skilled in field of the invention, and anyhow their definitions can be found in manuals like Gait, M. J. et al, 1984.
STATE OF THE ART
In general, applications that utilize synthetic oligonucleotides are of two kinds: those requiring the use of known sequence oligonucleotides, and those requiring the use of oligonucleotides with an at least partly degenerated or random sequence.
As for the first group of applications, the usual synthesis methods are based on the principle of building the polynucleotide condensing mononucleotides one at the time, starting from the first at the 3′-terminus, and choosing each mononucleotide for every reaction cycle so as to synthetize a polynucleotide with a desired and unambiguous sequence.
As for the second group of applications, the synthesis follows the same modalities, but in the positions along the sequence where one needs to insert variability the synthetic cycle goes on using mixtures of two or more different monomers. In every cycle oligonucleotide mixtures differing in the monomer added to the 5′-terminus are thus created. For instance, if in a cycle 4 different mononucleotides are employed as monomers, a mixture containing 4 different polynucleotides differing among themselves only for the last nucleotide inserted is obtained. If a synthetic cycle of the same kind is repeated, a mixture of 16 polynucleotides that differ in the last two inserted nucleotides is obtained, and so on.
In general, applications utilizing synthetic polynucleotides provide for a direct or indirect insertion of said polynucleotides in genetic material that will be translated into polypeptides in a certain living organism (in vitro translation seldom occurs). As it is known, DNA-translating genetic code is partly degenerated, i.e. as the 64 possible codons formed by groups of three nucleotides code for 20 amino acids only (plus three terminating or stop signals), more than one codon code for a single amino acid.
Oligonucleotides having an at least partly random sequence as aforedescribed (where for random sequence polynucleotide is meant a more or less complex mixture of polynucleotides having different sequences), code for random sequence peptides (i.e. for a mixture of peptides, each peptide being coded by one or more polynucleotides).
In fact, genetic code degeneracy en
Giesser Joanne M.
Istituto di Ricerche di Molecolare P. Angeletti S.p.A.
Lu Frank
Tribble Jack L.
Whisenant Ethan
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