Patent ID: 7853408

Claim:
Method for the design of oligonucleotides that can be used in molecular biology procedures, for identification of a metagenomic sample wherein it comprises the steps of (a) building a reference sequence database from a metagenomic sample; (b) selecting a subset of sequences (target sequences) corresponding to target organisms, according to taxonomic classification to build a design database; (c) selecting candidate oligonucleotides from the target sequences in the design database, by considering all subsequences of a defined length, between 18 and 50 nucleotides, present in the target sequences and their reverse-complementary sequences; (d) depurating these candidate oligonucleotides according to hybridization specificity and thermodynamic stability criteria, wherein the thermodynamic stability criteria are: i. only sequences forming a secondary structure with a Gibbs free energy difference threshold of at least −7 kcal/mole are considered valid; and ii. only sequences hybridizing with a copy of itself with a Gibbs free energy difference threshold of at least −7 kcal/mole are considered valid; and wherein the following criteria of specificity in hybridization are considered: iii. oligonucleotides designed according to taxonomic specificity are selected by looking for the designed oligonucleotide in the database of step (a) and registering the number of sequences belonging to each taxonomical group to which said oligonucleotide hybridizes in a list; or iv. oligonucleotides designed according to oligonucleotide sensitivity under consideration with respect to the group of target sequences are selected, wherein the sensitivity corresponds to a ratio between target sequences that were found and target sequences that exist in the design database; or v. oligonucleotides designed according to oligonucleotide selectivity under consideration with respect to the group of target sequences are selected, wherein the selectivity corresponds to a proportion of target sequences that were found belonging to the target taxon; or vi. oligonucleotides designed according to a product of the sensitivity multiplied by the selectivity of the oligonucleotide under consideration with respect to the group of target sequences, being selected those oligonucleotides where the product is higher than 50% of a maximum value among all of the oligonucleotides under consideration; (e) obtaining the list of oligonucleotides that fulfill requirements of step (d); (f) producing materially, by chemical synthesis, the designed oligonucleotides; and (g) selecting the oligonucleotides which comply with the requirements of the desired process.