Abstract:
A method of designing oligonucleotides useful in molecular biology techniques as PCR primers or in other techniques as identification and/or quantification probes is disclosed. The method permits designing specific oligonucleotides for the identification of a determined sequence in a metagenomic sample. The method includes selecting or constructing a database of reference sequences, selecting a subset of sequences belonging to target organisms, selecting candidate oligonucleotides from such sequences, depurating these candidate oligonucleotides according to hybridization specificity and thermodynamic stability criteria, obtaining a list of designed oligonucleotides that fulfill the hybridization specificity and thermodynamic stability criteria, producing materially by chemical synthesis the designed oligonucleotides, and selecting the oligonucleotides which comply with the requirements of the desired process.

Description:
FIELD OF THE INVENTION 
     The present invention discloses a method for the design of oligonucleotides useful in molecular biology techniques as PCR primers or in other techniques as identification and/or quantification probes. Specially, a method is disclosed to design specific oligonucleotides for the identification of a determined sequence in a metagenomic sample. 
     BACKGROUND OF THE INVENTION 
     Many methods in molecular biology require the use of short DNA sequences (oligonucleotides) satisfying given physicochemical and biological requirements to assess the presence of a certain organism or group of organisms. Among these methods, fluorescent in situ hybridization (FISH), denaturing gradient gel electrophoresis (DGGE), conjugation with specific markers, like detection or quantification probes for certain microorganisms, genes or sequences, and polymerase chain reaction (PCR), where two oligonucleotides are used as primers for the reaction, could be mentioned. This invention could be applied in said cases or in other cases wherein specific oligonucleotides are required. 
     Usually, oligonucleotides are artificially synthesized according to the description of their composing bases. The determination of the specific sequences that are suitable for each particular procedure is called “oligonucleotide design”. According to the involved procedure, certain thermodynamic restrictions could limit the set of valid oligonucleotides. Oligonucleotides resulting from this design procedure will be completely determined by the nucleotide sequences used in their synthesis, which could be characterized as words having finite length in the alphabet {A, C, T, G}. 
     Traditional oligonucleotide design methods, among which Primer3 (Rozen S., Skaletsky, H. (2000). Primer3 on the WWW for general users and for biologist programmers. In: Krawetz S, Misener S (eds) Bioinformatics Methods and Protocols: Methods in Molecular Biology. Humana Press, Totowa, N.J., pp 365-386) can be mentioned, allow the design of oligonucleotides pairs or primers for PCR amplification, validating a series of thermodynamic requirements. However, these methods only allow the design of oligonucleotides for a particular sequence, not considering the case where many sequences from different organisms are to be recognized. The traditionally used method in this case requires performing a multiple alignment of all the sequences that are to be recognized, by means of a computer program as CLUSTALW (Higgins D., Thompson J., Gibson T. (1994). CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22:4673-4680). This alignment allows the determination of conserved regions among all the sequences to be recognized and therefore the design of oligonucleotides within these regions. However, the performance of these alignments is expensive and could be prohibitive when the number of sequences is large. Moreover, multiple alignments require the determination of penalty parameters derived from some evolutionary model of the sequences. The result depends on the values chosen for these penalties and may not be robust when confronted to small changes in these values. 
     Among other methods for oligonucleotide design that have been developed in the last years, document US2003097223 (Nakae &amp; Ihara, 22/05/2003) could be mentioned, for instance, which protects a new primer design method. This method automatically designs primer pairs and then these primer pairs are selected according to certain requirements, namely oligonucleotide length, GC content percentage and Tm (melting temperature). Besides the basic aspects in primer design, well-known for someone skilled in the art, the method of the present invention considers a thermodynamic analysis of the designed primers, which adds an advantage over the method described in US2003097223 as the stability of the designed primers is guaranteed, improving the success probabilities of the use of said primers. Another different aspect between the former document and the invention herein disclosed is the fact that said document points to the finding of primers useful for many exons of a genome, whereas in one aspect of the invention all the microorganisms belonging to certain taxon are to be amplified; this fact constitutes a difference by itself, but the strategy used in both cases to find primers or oligonucleotides that could recognize more than one template is also different in both cases: in document US2003097223 a plurality of primers is designed (indicated as step 701) using bioinformatics means from a data base comprising different exons (step 700), and then PCR amplified DNA fragments are analyzed together with the designed primers, and primers amplifying target exons are empirically determined. Inversely, in the present invention primers present in the maximum number of target sequences are identified from the design database (which includes the target sequences) and primers to be synthesized and used are chosen based on this information. 
     Another document belonging to a related field in the art is the paper of Wang and Seed: “A PCR primer bank for quantitative gene expression analysis”, Nucleic Acids Research, 2003, vol. 31, No. 24 e154, where an algorithm is validated for the identification of specific transcription primers for PCR; the authors have created an online database with primers that fulfill said requirements for human and mice genes. The algorithm described by Wang and Seed significantly differs form the method proposed in the present invention, firstly because it does not contemplate the possibility of choosing an oligonucleotide or a primer pair common to a determined taxon, but specific primers are chosen for only one target sequence, and secondly because in the oligonucleotide selection procedure ΔG is evaluated only for the last 5 residues at the 3′ end of the molecule and the candidate is rejected when such value is less than −9 kcal/mole. In the present invention, ΔG is evaluated for all the candidate oligonucleotides and the selection criteria is much stringent, as preferentially only oligonucleotides having ΔGh min  equal to −1.5 kcal/mole (ΔG for hairpin formation) are selected. In order to predict the formation of hairpins in the referred paper, sequence auto-complementarity is evaluated and only 5 non-contiguous matches are allowed. In the same way, to avoid the formation of primer dimers the presence of complementary sequences in 4 residues at the 3′ end of the molecule in the same primer (to avoid dimers) and in the other primer (to avoid cross-reactivity) is evaluated. In the present invention, secondary structure formation is faced in a different and more efficient way than the simple sequence complementarity comparison; in this case, differences in Gibbs free energies are evaluated for all possible conformations and the probability of each selected oligonucleotide to form secondary structures is determined based on the most stable conformation. 
     As can be appreciated, the method of the invention shows indisputable technical advantages over other existent methods in the state of the art. 
     In summary, up to this date no oligonucleotide design method has been disclosed being fast and economical and allowing the design of specific oligonucleotides for a target sequence when said sequence is part of a metagenomic sample or allowing the design of oligonucleotides that simultaneously recognize various sequences belonging to different organisms. 
     In this disclosure, said problems of the existing technique have been solved, creating a method for the design of specific oligonucleotides for a given sequence or group of sequences, that considers not only the information of the genetic material to be identified but also the information of all the genetic material that could be present in a metagenomic sample over which the method will be applied. 
     Another common problem in the field of oligonucleotide design is the fact that even when an oligonucleotide meeting the required specificity could be available, in practice of molecular biology procedures said oligonucleotide is not efficient. Explanations for this inefficiency are formation of secondary structures within the oligonucleotide sequence (hairpins) or auto-hybridization, which decreases the active concentration of the oligonucleotide in the reaction mix. In the case of PCR technique, where an oligonucleotide pair is simultaneously used, a cross-hybridization between both oligonucleotides could be possible, besides auto-hybridization and hairpin formation, which also sequesters oligonucleotides in the reaction mix and makes said reaction inefficient. 
     In order to overcome this technical problem, the method of the invention includes a step wherein the designed oligonucleotides are thermodynamically evaluated to discard formation of hairpins, auto-hybridization or cross-hybridization between two primers. For each of these situations, Gibbs free energy differences are calculated for all the possible conformations, the most stable conformation being selected; if said most stable conformation has a ΔG value less than a certain threshold, said oligonucleotide is discarded, thus guaranteeing the availability of the designed oligonucleotides. 
     Thus, the method of the present invention allows solving all the problems existing in the field of oligonucleotide design for Molecular Biology techniques. 
     SUMMARY OF THE INVENTION 
     As previously described, the present invention discloses a method that can be used to identify one DNA or RNA sequence or one specific group of DNA or RNA sequences from a complex biological sample. 
     Diverse molecular biology methods require the presence of short DNA sequences, called oligonucleotides, that are artificially synthesized from a description of their composing bases. 
     The oligonucleotide design method comprises the selection or construction of a database of reference sequences, the selection of a subset of sequences belonging to target organisms, the selection of candidate oligonucleotides from such sequences, the depuration of these candidate oligonucleotides according to hybridization specificity and thermodynamic stability criteria, which allows to obtain a list of designed oligonucleotides and, optionally, the sorting of such oligonucleotides according to their taxonomical specificity. 
     The extension of this method to the case in which oligonucleotides pairs are required is also disclosed, as could be the case of polymerase chain reaction (PCR) procedures. This method variant comprises the construction or selection of a database of reference sequences, the selection of a subset of sequences belonging to target organisms, the selection of two sets of candidate oligonucleotides from such sequences, the depuration of each set of candidate oligonucleotides according to hybridization specificity and thermodynamic stability criteria, the elaboration of a list of oligonucleotides pairs or primers formed by one element from each set that satisfy physical and thermodynamic requirements and the sorting of such oligonucleotides pairs according to taxonomical specificity. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  shows the results of a PCR performed with oligonucleotides pairs or primers designed using the method of the invention to carry out a specific PCR for  A. thiooxidans  amplifying 16S rDNA from 5 samples of  A. ferrooxidans  and 2 samples of  A. thiooxidans ; the content of each lane is specified in the Examples section, in Table 5. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     Oligonucleotide Design. 
     The method for the design of oligonucleotides herein described takes a database of DNA or RNA sequences as an input. Depending on experimental requirements being considered, these sequences may be complete genomes or fragments from each genome. For instance, all known sequences of a given gene or genomic region could be considered. In a preferred embodiment of the present invention, a database designed by us is considered, which contains all known sequences of gene 16S. A requirement to be met by the database under use is that every sequence must have been taxonomically classified. An example of database that can be used as input with the method of this invention is GenBank, from NCBI (Benson, D. A., Boguski, M. S., Lipman, D. J., Ostell, J. (1997). GenBank. Nucleic Acids Res. Jan 1; 25(1):1-6). This selected or constructed database is called “evaluation database”. From this database, the sequence subset corresponding to the organism(s) to be identified is extracted. This subset is called “design database”. 
     In some cases, considering the fact that partial sequences of the target genes could be found in public databases, it is convenient to normalize the relative positions within each sequence, and so each sequence in the design database is optimally aligned to a reference sequence, which may be a gene that is homologous to the analyzed one, using the Needleman-Wunsch algorithm (Needleman, S. B., Wunsch, C. D. (1970).  A general method applicable to the search for similarities in the amino acid sequence of two proteins . J Mol Biol. Mar; 48(3):443-53). In said case, these aligned sequences form the design database. 
     Once the design database has been defined, the oligonucleotide set therein contained is established. This oligonucleotide set is built considering each subsequence, hereinafter called ‘words’, that has a defined size (typically between 18 and 50 letters) and is contained in each subsequence and also in the subsequences that are complementary reverse to them. The words that are present more than once in some sequence are discarded, considering also a number of substitutions within the word, which typically could be up to 15% of the letters contained in the word. For instance, in a 20-letter word, 15% corresponds to 3 substitutions, so if a word having length 20 is coincident in 17 or more letters with another word of the same sequence, both words are discarded. This procedure is efficiently performed if the hereinbelow described algorithm is followed, taking as inputs the design database, the size of the oligonucleotides being designed (n) and the maximum number of allowed letter substitutions (u). The selection of candidate oligonucleotides is performed by taking into account all subsequences of defined length that are present in the target sequences and their corresponding reverse-complementary sequences. 
     
       
         
               
               
             
               
               
             
           
               
                   
               
               
                   
                 Algorithm 1 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 For each sequence S i  in the design database 
               
               
                   
                  Consider each word P ij  being a previously unseen subsequence of S i  with length n 
               
               
                   
                  For each sequence S k , C k  is defined as the number of times the oligonucleotide appears 
               
               
                   
                 within each candidate sequence, wherein subscript k is the sequence number. 
               
               
                   
                  C k  ← 0 
               
               
                   
                  For each word P kl  being a subsequence of S k  with length n 
               
               
                   
                   If word P kl  coincides with P ij  in at least n-u letters 
               
               
                   
                   C k  ← C k  +1 
               
               
                   
                   If word P kl  coincides with P ij  in exactly n letters 
               
               
                   
                   Mark word P kl  as previously seen 
               
               
                   
                   Remember that P ij  hybridizes with S k  on strand +1 
               
               
                   
                  For each word P′ kl  being a reverse-complementary sequence of P kl   
               
               
                   
                   If word P′ kl  coincides with P ij  in at least n-u letters 
               
               
                   
                   C k  ← C k  +1 
               
               
                   
                   If word P′ kl  coincides with P ij  in exactly n letters 
               
               
                   
                   Mark word P kl  as previously seen 
               
               
                   
                   Remember that P ij  hybridizes with S k  on strand −1 
               
               
                   
                  If C k  is greater than 1 
               
               
                   
                   Mark word P ij  as discarded 
               
               
                   
                  If P ij  is not discarded 
               
               
                   
                  Print word P ij  as oligonucleotide candidate on strand +1 
               
               
                   
                  Consider each word P′ ij  being a previously unseen reverse-complementary sequence of P ij    
               
               
                   
                  For each sequence S k   
               
               
                   
                  C k  ← 0 
               
               
                   
                  For each word P kl  being a subsequence of S k  with length n 
               
               
                   
                   If word P kl  coincides with P′ ij  in at least n-u letters 
               
               
                   
                   C k  ← C k  +1 
               
               
                   
                   If word P kl  coincides with P′ ij  in exactly n letters 
               
               
                   
                   Mark word P kl  as previously seen 
               
               
                   
                   Remember that P′ ij  hybridizes with S k  on strand +1 
               
               
                   
                  For each word P′ kl  being a reverse-complementary sequence of P kl   
               
               
                   
                   If word P′ kl  coincides with P′ ij  in at least n-u letters 
               
               
                   
                   C k  ← C k  +1 
               
               
                   
                   If word P′ kl  coincides with P′ ij  in exactly n letters 
               
               
                   
                   Mark word P′ kl  as previously seen 
               
               
                   
                   Remember that P′ ij  hybridizes with S k  on strand −1 
               
               
                   
                  If C k  is greater than 1 
               
               
                   
                   Mark word P′ ij  as discarded 
               
               
                   
                  If P′ ij  is not discarded 
               
               
                   
                  Print word P′ ij  as oligonucleotide candidate on strand −1 
               
               
                   
               
             
          
         
       
     
     Each word or subsequence generated by the described algorithm is called “candidate oligonucleotide”. In this first attempt a large quantity of candidate oligonucleotides is obtained, which are submitted to the selection criteria of the method. 
     These candidates are then evaluated by their thermodynamic stability. The first evaluation is the determination of the Gibbs free energy for the smallest energy secondary structure. This means that Gibbs free energy difference is calculated for all the spatial conformations where the oligonucleotide hybridizes with itself, until the structure with the smallest energy difference, i.e. the most stable structure, is found. If this value, which is called ΔGh min  as it defines the Gibbs free energy difference for hairpin formation of the oligonucleotide, is smaller than a threshold value, defined in a first attempt as the best quartile, the candidate oligonucleotide is discarded. If it is desired to reduce even more the number of candidate oligonucleotides, a larger ΔGh min  could be selected, which may be, e.g., −7 kcal/mole. The ΔGh min  threshold value preferably used according to the invention is −1.5 kcal/mole. 
     For certain procedures where designed oligonucleotide concentration would be too high, as in polymerase chain reaction (PCR) or fluorescent in situ hybridization (FISH), a second validation should be performed, which requires the evaluation of the smallest Gibbs free energy of all the structures formed by two copies of the candidate oligonucleotide. Analogously, if this energy does not surpass a threshold value for ΔGd min , which defines the Gibbs free energy difference for the formation of oligonucleotide dimers, the oligonucleotide is discarded. In a first approach, the threshold is defined as the best quartile and, if a stricter bound for the oligonucleotide number is desired, a larger ΔGd min  can be selected. The ΔGd min  threshold value preferentially used according to this invention is −7 kcal/mole. 
     Methods to calculate these minimal energies are well known and have been described in literature, for instance: 
     Bommarito S., Peyret N., SantaLucia J. Jr. (2000).  Thermodynamic parameters for DNA sequences with dangling ends . Nucleic Acids Res. May 1; 28(9):1929-34. D. H. Mathews, J. Sabina, M. Zuker &amp; D. H. Turner. (1999)  Expanded Sequence Dependence of Thermodynamic Parameters Improves Prediction of RNA Secondary Structure . J. Mol. Biol. 288, 911-940. 
     M. Zuker. (2003)  Mfold web server for nucleic acid folding and hybridization prediction . Nucleic Acids Res. 31 (13), 3406-15. 
     Ivo L. Hofacker, Walter Fontana, Peter F. Stadler, L. Sebastian Bonhoeffer, Manfred Tacker, and Peter Schuster (1994).  Fast Folding and Comparison of RNA Secondary Structures . Monatsh. Chem.  125: 167-188. 
     All the oligonucleotides that have not been discarded in the former stage are oligonucleotides designed by the method and are useful in molecular biology procedures. 
     Identification of Taxonomical Groups with Designed Oligonucleotides. 
     In identification procedures, it is desired to mark the presence of a specific taxonomical group in the sample. For this purpose, the evaluation database is analyzed looking for each oligonucleotide, registering the taxonomical group of the sequence in which the oligonucleotide appears. This operation generates, for each oligonucleotide, a table indicating the number of sequences belonging to each taxonomical group to which said oligonucleotide hybridizes. This table allows the calculation of two taxonomical specificity indexes given the target taxonomical group for each oligonucleotide, said indexes being described as follows: 
     Let N be the number of sequences belonging to the target taxonomical group that are present in the evaluation database. Let T be the number of sequences belonging to the target taxonomical group to which said oligonucleotide hybridizes; and let R be the total number of sequences to which said oligonucleotide hybridizes. We use “Sensitivity” to designate the percentage or ratio of target sequences effectively found. That is:
 
Sensitivity=T/N
 
     Analogously, we use “Selectivity” to designate the percentage or ratio of found sequences belonging to the target group. That is:
 
Selectivity=T/R
 
     For instance, if the target taxonomical group is  Escherichia coli , there are N=80 sequences in the evaluation database belonging to this species, and the oligonucleotide hybridizes to R=120 sequences, of which T=60 belong to  E. coli , then the Sensitivity of this oligonucleotide is
 
Sensitivity=T/N=60/80=0.75
 
     Whereas the Selectivity is
 
Selectivity=T/R=60/120=0.5
 
     In other words, the oligonucleotide allows the identification of 75% of the sequences belonging to  E. coli . Of all the recognized sequences, 50% belong to  E. coli.    
     Most suitable oligonucleotides for identification procedures should simultaneously maximize both indexes. The following step in the method of this invention is the selection of the oligonucleotide with the largest Sensitivity and Selectivity, simultaneously. This can be achieved by, for example, obtaining the product of both indexes for each oligonucleotide and choosing the largest value thus obtained; this product is called “Rate”. 
     The following algorithm describes the procedure to calculate these indexes for an oligonucleotide, represented as 0, as a function of the number of letters forming O (represented by n) and the maximum number of permitted substitutions, represented by u: 
     
       
         
               
             
               
               
             
               
               
             
               
               
             
               
               
             
               
               
             
               
               
             
               
               
             
               
               
             
               
               
             
           
               
                   
               
               
                 Algorithm 2 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 Let be T ← 0 
               
               
                   
                 Let be N ← 0 
               
               
                   
                 Let be R ← 0 
               
               
                   
                 For each sequence S i  in the reference database 
               
             
          
           
               
                   
                 If S i  belongs to the target taxonomical group 
               
             
          
           
               
                   
                 N ← N+1 
               
             
          
           
               
                   
                 For each word P ij  being a subsequence of S i  with length n 
               
             
          
           
               
                   
                 If P ij  coincides with O in more than n-u letters 
               
             
          
           
               
                   
                 R ← R+1 
               
               
                   
                 If S i  belongs to the target taxonomical group 
               
             
          
           
               
                   
                 T ← T+1 
               
             
          
           
               
                   
                 Finally, 
               
             
          
           
               
                   
                 Sensitivity ← T/N 
               
               
                   
                 Selectivity ← T/R 
               
               
                   
                 Rate ← T 2 /(N-R) 
               
               
                   
               
             
          
         
       
     
     The oligonucleotides designed and selected according to this method are useful in molecular biology procedures intended to determine the presence of a target taxonomical group in a complex sample. Generally, they are produced by chemical synthesis and could be labeled by any known labeling technique, e.g. radioactive, fluorescent or chemiluminiscent labeling. 
     Design of Oligonucleotides Pairs or Primers. 
     Certain types of molecular biology procedures require the simultaneous presence of many different oligonucleotides. For instance, polymerase chain reaction (PCR) requires the presence of two oligonucleotides that satisfy certain requirements. The subject method of this invention is complemented in this case by the following steps. 
     Further to the abovementioned elements, this complementary stage requires the definition of a maximum and minimum size for the desired PCR product and a limit for the melting temperature (Tm) difference between both oligonucleotides. 
     To start, oligonucleotides designed according to Algorithm 1 are considered. 
     For each sequence in the design database, oligonucleotides pairs or primers formed by oligonucleotides that hybridize to the sequence are considered, in such a way that the first oligonucleotide hybridizes to the forward strand and the second oligonucleotide hybridizes to the reverse strand. The “amplification size” is calculated as the difference between the hybridization positions of the second oligonucleotide minus the first one. Pairs of oligonucleotides having amplification sizes outside the pre-established range are discarded. 
     For each oligonucleotide, Tm is calculated using the method described by Le Novére N. (2001).  MELTING, computing the melting temperature of nucleic acid duplex . Bioinformatics. 2001 Dec; 17(12):1226-7. 
     Pairs of oligonucleotides having a melting temperature difference over the pre-established temperature difference, which is preferably less than 4° C., are discarded. 
     Once an oligonucleotide pair list fulfilling the established requirements is obtained using the described method, the thermodynamic stability of the oligonucleotide pair is evaluated by determining the minimal energy structure formed by both oligonucleotides when hybridizing each other. If this energy, which is called ΔGx min  as it defines the Gibbs free energy difference for cross-hybridization between both oligonucleotides, is smaller than a threshold value, defined in a first attempt as the best quartile, the oligonucleotide is discarded. In all cases, such threshold should not be lower than −12 kcal/mole. 
     The method used to calculate ΔGx min  is described in M. Zuker. (2003)  Mfold web server for nucleic acid folding and hybridization prediction . Nucleic Acids Res. 31 (13), 3406-15. 
     Oligonucleotides pairs or primers that fulfill size restrictions for the amplification product, melting temperature difference restrictions and thermodynamic stability restrictions should be evaluated according to their taxonomical specificity. The abovementioned Selectivity and Sensitivity parameters are evaluated but for each oligonucleotide pair member. An oligonucleotide pair is considered to hybridize to a target sequence if both oligonucleotides hybridize to said sequence. That is, the set of sequences to which the oligonucleotide pair hybridizes is the intersection of the sets of sequences to which each oligonucleotide hybridizes. 
     Having been determined the set of sequences to which the oligonucleotide pair hybridizes, the corresponding Selectivity and Sensitivity indexes are calculated, and pairs that maximize both criteria are selected for the molecular biology procedure. 
     The following described algorithm allows the identification of oligonucleotides pairs or primers that satisfy the requirements described above. It should be taken into account that for each of them the strand to which it hybridizes (1 or −1) and the melting temperature, called Tm, has already been determined in the oligonucleotide design stage. 
     
       
         
               
             
               
             
               
               
             
               
               
             
               
               
             
               
               
             
               
               
             
               
               
             
               
               
             
               
               
             
               
               
             
               
               
             
               
               
             
               
               
             
               
             
           
               
                   
               
               
                 Algorithm 3 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 For each sequence S i  in the design database 
               
             
          
           
               
                   
                 For each oligonucleotide F ij  that hybridizes to S i  at 
               
               
                   
                 position j of strand 1 
               
             
          
           
               
                   
                 For each oligonucleotide R ik  that hybridizes to S i   
               
               
                   
                 at position k of strand −1 
               
             
          
           
               
                   
                 Size ← k-j (size of the amplified region) 
               
               
                   
                 If Size is within the specified range 
               
             
          
           
               
                   
                 If Tm(F ij ) is different from Tm(R ik ) in 
               
               
                   
                 no more than 2 degrees 
               
               
                   
                 ΔGd ← Heterodimer free energy 
               
               
                   
                 If ΔGd &gt; ΔGd min   
               
             
          
           
               
                   
                 Let be T ← 0 
               
               
                   
                 Let be N ← 0 
               
               
                   
                 Let be R ← 0 
               
             
          
           
               
                   
                 For each sequence S m  in the reference database 
               
             
          
           
               
                   
                 If S m  belongs to the target taxonomical group 
               
             
          
           
               
                   
                 N ← N+1 
               
             
          
           
               
                   
                 If S m  simultaneously contains F ij  and R ik   
               
             
          
           
               
                   
                 R ← R+1 
               
               
                   
                 If S m  belongs to the target taxon 
               
             
          
           
               
                   
                 T ← T+1 
               
             
          
           
               
                   
                 Sensitivity ← T/N 
               
               
                   
                 Selectivity ← T/R 
               
               
                   
                 Rate ← T 2 /(N·R) 
               
               
                   
                 Print F ij , R ik , Sensitivity, Selectivity, Rate 
               
             
          
           
               
                 The oligonucleotide pair that maximizes “Rate” is selected 
               
               
                   
               
             
          
         
       
     
     The oligonucleotide pair that maximizes the “Rate” parameter is selected. The oligonucleotides pairs or primers designed and selected according to this method are useful in molecular biology procedures, such as PCR, intended to determine the presence of a target taxonomical group in a complex sample. 
     EXAMPLES 
     Example 1 
     Design of a Specific Oligonucleotide for Bacteria Belonging to  Leptospirillum  Genus. 
     A new database was obtained with data comprising only 16S sequences selected from the public NCBI GenBank database. This new database is the “evaluation database”. 
     All sequences that come from bacteria belonging to  Leptospirillum  genus, 44 sequences in this case, were selected to be the “design database”. 
     The set of 20-letter oligonucleotides that are present in each of the sequences was determined, discarding those sequences appearing more than once within each sequence, considering up to 3 substitutions, using Algorithm 1. These oligonucleotides are the “candidate oligonucleotides”, which were evaluated according to their thermodynamic stability using the algorithm described in M. Zuker. (2003) Mfold web server for nucleic acid folding and hybridization prediction, Nucleic Acids Res. 31 (13), 3406-15. All candidate oligonucleotides with ΔGh values lower than −1.5 kcal/mole or with ΔGd values lower than −7 kcal/mole were discarded. This analysis provided a total of 14785 oligonucleotides that were present in at least one of the 44 sequences in the design database. None of them is present in all the design sequences. Oligonucleotides present in most of the sequences were considered. This reduced the list to 12 oligonucleotides, which are the oligonucleotides designed by the method and have the following structures: 
     
       
         
               
             
               
               
               
             
               
             
               
               
               
             
               
             
               
               
               
             
               
             
               
               
               
             
               
             
               
               
               
             
               
             
               
               
               
             
               
             
               
               
               
             
               
             
               
               
               
             
               
             
               
               
               
             
               
             
               
               
               
             
               
             
               
               
               
             
               
             
               
               
               
             
           
               
                 (SEQ ID NO: 1) 
               
             
          
           
               
                   
                 Oligonucleotide 1: 
                 TACAGACTCTTTACGCCCAG 
               
               
                   
               
             
          
           
               
                 (SEQ ID NO: 2) 
               
             
          
           
               
                   
                 Oligonucleotide 2: 
                 CTACAGACTCTTTACGCCCA 
               
               
                   
               
             
          
           
               
                 (SEQ ID NO: 3) 
               
             
          
           
               
                   
                 Oligonucleotide 3: 
                 CCTACAGACTCTTTACGCCC 
               
               
                   
               
             
          
           
               
                 (SEQ ID NO: 4) 
               
             
          
           
               
                   
                 Oligonucleotide 4: 
                 ACCTACAGACTCTTTACGCC 
               
               
                   
               
             
          
           
               
                 (SEQ ID NO: 5) 
               
             
          
           
               
                   
                 Oligonucleotide 5: 
                 CACCTACAGACTCTTTACGC 
               
               
                   
               
             
          
           
               
                 (SEQ ID NO: 6) 
               
             
          
           
               
                   
                 Oligonucleotide 6: 
                 CCACCTACAGACTCTTTACG 
               
               
                   
               
             
          
           
               
                 (SEQ ID NO: 7) 
               
             
          
           
               
                   
                 Oligonucleotide 7: 
                 CTGGGCGTAAAGAGTCTGTA 
               
               
                   
               
             
          
           
               
                 (SEQ ID NO: 8) 
               
             
          
           
               
                   
                 Oligonucleotide 8: 
                 TGGGCGTAAAGAGTCTGTAG 
               
               
                   
               
             
          
           
               
                 (SEQ ID NO: 9) 
               
             
          
           
               
                   
                 Oligonucleotide 9: 
                 GGGCGTAAAGAGTCTGTAGG 
               
               
                   
               
             
          
           
               
                 (SEQ ID NO: 10) 
               
             
          
           
               
                   
                 Oligonucleotide 10: 
                 GGCGTAAAGAGTCTGTAGGT 
               
               
                   
               
             
          
           
               
                 (SEQ ID NO: 11) 
               
             
          
           
               
                   
                 Oligonucleotide 11: 
                 GCGTAAAGAGTCTGTAGGTG 
               
               
                   
               
             
          
           
               
                 (SEQ ID NO: 12) 
               
             
          
           
               
                   
                 Oligonucleotide 12: 
                 CGTAAAGAGTCTGTAGGTGG 
               
             
          
         
       
     
     Example 2 
     Identification of Bacteria Belonging to  Leptospirillum  Genus in a Metagenomic Sample 
     The reference database was searched looking for said 12 oligonucleotides designed in Example 1, and the following Sensitivity and Selectivity values were obtained: 
     
       
         
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE 1 
               
               
                   
               
               
                 Oligo 
                 N 
                 T 
                 R 
                 Sensitivity 
                 Selectivity 
                 Rate 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 1 
                 54 
                 37 
                 44 
                 84.1% 
                 68.5% 
                 57.6% 
               
               
                 2 
                 68 
                 37 
                 44 
                 84.1% 
                 54.4% 
                 45.8% 
               
               
                 3 
                 66 
                 37 
                 44 
                 84.1% 
                 56.1% 
                 47.1% 
               
               
                 4 
                 58 
                 37 
                 44 
                 84.1% 
                 63.8% 
                 53.6% 
               
               
                 5 
                 57 
                 37 
                 44 
                 84.1% 
                 64.9% 
                 54.6% 
               
               
                 6 
                 56 
                 37 
                 44 
                 84.1% 
                 66.1% 
                 55.6% 
               
               
                 7 
                 54 
                 37 
                 44 
                 84.1% 
                 68.5% 
                 57.6% 
               
               
                 8 
                 68 
                 37 
                 44 
                 84.1% 
                 54.4% 
                 45.8% 
               
               
                 9 
                 66 
                 37 
                 44 
                 84.1% 
                 56.1% 
                 47.1% 
               
               
                 10 
                 58 
                 37 
                 44 
                 84.1% 
                 63.8% 
                 53.6% 
               
               
                 11 
                 57 
                 37 
                 44 
                 84.1% 
                 64.9% 
                 54.6% 
               
               
                 12 
                 56 
                 37 
                 44 
                 84.1% 
                 66.1% 
                 55.6% 
               
               
                   
               
               
                 N = number of sequences belonging to the target taxonomical group that are present in the evaluation database. 
               
               
                 T = number of sequences belonging to the target taxonomical group to which said oligonucleotide hybridizes; and 
               
               
                 R = total number of sequences to which said oligonucleotide hybridizes. 
               
             
          
         
       
     
     According to these results, oligonucleotides 1 and 7 were selected as best candidates. Both were synthesized, fluorescently labeled with Cy5 and used as probes to identify bacteria belonging to  Leptospirillum  genus in a metagenomic sample by using the FISH technique. To assess that what was detected corresponds only to  Leptospirillum , controls were carried out with pure  Leptospirillum ferrooxidans  cultures, and a specific detection was found. 
     Example 3 
     Design of a Specific Oligonucleotide Pair for Bacteria Belonging to  Sulfobacillus thermosulfidooxidans  Species 
     In the “evaluation database” obtained in Example 1, existing sequences for  Sulfobacillus thermosulfidooxidans  bacteria were selected, 8 sequences in this case, which form the “design database”. 
     Two sets of oligonucleotides that have 19 to 21 letters present in each of the sequences were determined, discarding those sequences appearing more than once within each sequence, considering up to 3 substitutions, using Algorithm 1. All oligonucleotides with a ΔGh value lower than −1.5 kcal/mole and with a ΔGd value lower than −7 kcal/mole were discarded. The first set considers all sub-sequences with 19 to 21 nucleotides in the target sequences and the second set considers the corresponding reverse-complementary sequences. Then, oligonucleotides pairs or primers that have hybridization sites with 200-500 nucleotides between them were determined. This primer pairs were evaluated according to their thermodynamic stability using the criteria described in M. Zuker. (2003)  Mfold web server for nucleic acid folding and hybridization prediction . Nucleic Acids Res. 31 (13), 3406-15, and all pairs having a cross hybridization energy ΔGx min  lower than −12 kcal/mole were discarded. This analysis provided a total of 237,223 oligonucleotides pairs or primers that were present in the 8 sequences in the design database. These 237,223 oligonucleotides pairs or primers constitute the “candidate oligonucleotides pairs or primers” designed by the method. 
     For each of these pairs the taxonomical specificity was evaluated in terms of their “Sensitivity”, “Selectivity” and “Rate” indexes. The first 5 primer pairs selected according to these criteria are shown in Table 2 
     
       
         
               
               
               
             
           
               
                 TABLE 2 
               
               
                   
               
               
                   
                 Sense Primer 
                 Antisense Primer 
               
               
                   
               
             
             
               
                 Pair 1 
                 GCTTGGCAACAGGCGCTCA 
                 GGCTTCCTCCGTCGGTACCG 
               
               
                   
                 (SEQ ID NO: 13) 
                 (SEQ ID NO: 14) 
               
               
                   
               
               
                 Pair 2 
                 TGAGTGGGGGATATCGGGCC 
                 TTTGCAGGGGCTTCCTCCGT 
               
               
                   
                 (SEQ ID NO: 15) 
                 (SEQ ID NO: 16) 
               
               
                   
               
               
                 Pair 3 
                 AGGCGCTCACAGGGGAGCTC 
                 GCGGCTGCTGGCACGTAGTT 
               
               
                   
                 (SEQ ID NO: 17) 
                 (SEQ ID NO: 18) 
               
               
                   
               
               
                 Pair 4 
                 GGTGAGGAACACGTGAGTG 
                 CCGGAGGCTTAAAACCGCT 
               
               
                   
                 (SEQ ID NO: 19) 
                 (SEQ ID NO: 20) 
               
               
                   
               
               
                 Pair 5 
                 CGGGCTGTGAGTGGGGGAT 
                 GGGGCTTCCTCCGTCGGTA 
               
               
                   
                 (SEQ ID NO: 21) 
                 (SEQ ID NO: 22) 
               
               
                   
               
             
          
         
       
     
     Example 4 
     Other Results 
     Primer Design for Different Taxons. 
     The described method has been used for the design of many primer pairs specific for different taxons, like  Acidithiobacillus thiooxidans, Acidithiobacillus ferrooxidans, Leptospirillum  sp.,  Acidiphillum  sp. In Table 3 thermodynamic and specificity requirements are shown corresponding to 4 oligonucleotides pairs or primers that were designed using the method of the invention to perform a specific PCR for each microorganism indicated in said Table, namely  A. ferrooxidans, A. thiooxidans, Leptospirillum  sp. y  Acidiphillum  sp. These oligonucleotides are useful as PCR primers for said taxons. 
     Table 4 shows sequences of selected specific primers. 
     
       
         
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
             
               
             
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
             
               
             
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
             
               
             
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
             
               
             
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 3 
               
             
             
               
                   
               
               
                 Sense 
                 Antisense 
                 Size 
                 Dimer 
                 Specificity 
               
             
          
           
               
                 Primer 
                 T m   
                 ΔG d   
                 ΔG h   
                 Primer 
                 T m   
                 ΔG d   
                 ΔG h   
                 Min 
                 Max 
                 ΔG x   
                 Sensit 
                 Select 
                 Rate 
                 Org 
                 Sp 
               
               
                   
               
             
          
           
               
                   A. ferrooxidans  Primers 
               
             
          
           
               
                 TH674.5F 
                 62 
                 −3.44 
                 1.53 
                 TH1116.11R 
                 60 
                 −3.23 
                 0.57 
                 444 
                 444 
                 −9.98 
                 4.00 
                 100.00 
                 4.00 
                 1 
                 1 
               
             
          
           
               
                   A. thiooxidans  Primers 
               
             
          
           
               
                 TH1143.9F 
                 68 
                 −2.84 
                 −0.79 
                 TH1393.1R 
                 62 
                 −2.18 
                 0.36 
                 250 
                 250 
                 −7.53 
                 20.00 
                 100.00 
                 20.00 
                 1 
                 1 
               
             
          
           
               
                   Leptospirillum  sp. Primers 
               
             
          
           
               
                 LP1233.1F 
                 60 
                 −4.49 
                 1.07 
                 LP1472.1R 
                 58 
                 −4.88 
                 1.47 
                 241 
                 241 
                 −11.38 
                 18.18 
                 100.00 
                 18.18 
                 8 
                 8 
               
             
          
           
               
                   Acidiphillum  sp. Primers 
               
             
          
           
               
                 AP202.6F 
                 62 
                 −2.65 
                 1.40 
                 AP626.2R 
                 62 
                 −2.27 
                 −1.18 
                 373 
                 373 
                 −11.27 
                 2.63 
                 100.00 
                 2.63 
                 1 
                 1 
               
               
                   
               
             
          
         
       
     
     
       
         
               
               
             
               
               
               
             
           
               
                 TABLE 4 
               
               
                   
               
               
                 Primer 
                 Sequence 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 TH674.5F 
                 GAATTCCAGGTGTAGCGGTG 
                 (SEQ ID NO: 23) 
               
               
                   
               
               
                 TH1116.11R 
                 AACCGCTGCAACTAAGGACA 
                 (SEQ ID NO: 24) 
               
               
                   
               
               
                 TH1143.9F 
                 GGGACTCAGTGGAGACCGCC 
                 (SEQ ID NO: 25) 
               
               
                   
               
               
                 TH1393.1R 
                 GTGTGACGGGCGGTGTGTA 
                 (SEQ ID NO: 26) 
               
               
                   
               
               
                 LP129.4F 
                 GATCTGCCCTGGAGATGGGG 
                 (SEQ ID NO: 27) 
               
               
                   
               
               
                 LP381.1R 
                 CGTTGCTGCGTCAGGGTTG 
                 (SEQ ID NO: 28) 
               
               
                   
               
               
                 AP112.1F 
                 GGTGAGTAACGCGTAGGAA 
                 (SEQ ID NO: 29) 
               
               
                   
               
               
                 AP363.1R 
                 TCGCCCATTGTCCAATATT 
                 (SEQ ID NO: 30) 
               
               
                   
               
             
          
         
       
     
     Design of specific primers for  A. thiooxidans  useful in a metagenomic sample. 
     In other study, the method of the present invention was used to design specific primers for  Acidithiobacillus thiooxidans  to be used in a metagenomic sample. Primer pairs were designed using the described method and the 4 primer pairs having the best “Rate” indexes were selected. PCR tests were carried out using 16S rDNA from 5  Acidithiobacillus ferrooxidans  samples and 2  Acidithiobacillus thiooxidans  samples, which were amplified using each designed primer pair. 
     PCR protocol used was as follows:
         1. −95° C. for 5 minutes   2. −95° C. for 30 seconds   3. −62° C. for 30 seconds   4. −72° C. for 20 seconds   5. − go to step (2) 29 more times   6. −10° C. until tubes were removed       

       FIG. 1  shows PCR results, the lanes in  FIG. 1  having the following load: 
     
       
         
               
               
             
           
               
                 TABLE 5 
               
               
                   
               
               
                 Lane 
                 Sample 
               
               
                   
               
             
             
               
                 1 
                   A. ferrooxidans  DSM 16786 
               
               
                 2 
                   A. ferrooxidans  ATCC23270 
               
               
                 3 
                   A. ferrooxidans  DSM 14882 
               
               
                 4 
                   A. ferrooxidans  ATCC 19859 
               
               
                 5 
                   A. ferrooxidans  ATCC33020 
               
               
                 6 
                   A. thiooxidans  sp. 
               
               
                 7 
                   A. thiooxidans  DSM 504 
               
               
                 (-) 
                 Sterile water 
               
               
                   
               
             
          
         
       
     
     As can be appreciated in  FIG. 1 , all the designed primer pairs amplified  A. thiooxidans , whereas primer Pair C specifically amplifies  A. thiooxidans , with no amplification of  A. ferrooxidans . This means that Pair C could allow the specific determination of the presence of  A. thiooxidans  in a metagenomic sample, even when  A. ferrooxidans  is present in said sample, as is usually the case.