Method and compositions for identification of species in a sample using type II topoisomerase sequences

A method of identifying an organism in a sample, comprising the steps of:

• providing a specific nested primer composition for amplifying a segment of a nucleic acid encoding a protein selected from the group consisting of:
• type II topoisomerase enzymes and homologues of type II topoisomerase enzymes;
• the protein having a signature region having an amino acid sequence which differs among different species, the signature region further being flanked by consensus regions whose amino acid sequence does not substantially vary among different species;
• and the primer composition comprising a primer pair constructed to amplify a signature segment encompassed within the signature region;
• making an extract of DNA molecules from a sample;
• using the specific primer composition to selectively amplify the signature DNA region, to produce amplified DNA segments;
• and
• determining from the amplified DNA segments whether a particular specie(s) corresponding to the primer pair signature segment is present in the sample.

A method of using a database to identify at least one species of organism in a sample, comprising the steps of:

• providing a database including reference amino acid sequences and further containing one or more reference nucleotide sequences each comprising a residue nucleotide sequence of a signature region found in a type II DNA topoisomerase, the signature zone having a defined length which is similar in all species of a chosen group and varies significantly in residue nucleotide sequence among species in the chosen group, the signature region further being flanked by left and right consensus regions having respective residue nucleotide sequences which do not vary significantly among the species, said database further providing the respective variable portions denoted by numerals 6 and 16 of the sequences depicted in FIGS. 2, 5A and 5B (SEQ. ID Nos. 1-32);
• obtaining one or more nucleic acid segments from the sample, each said segment(s) derived from the signature region;
• determining a residue nucleotide sequence of at least one of the segment(s);
• converting the determined residue nucleotide sequence to an amino acid sequence;
• and
• comparing the converted amino acid sequence with said one or more reference amino acid sequences in the database to find a matching reference amino acid sequence, and thereby identify the sample organism.

A method of using a database to identify at least one species of organism in a sample, comprising the steps of:

• providing a database including SEQ. ID Nos. 33-100, said database further containing one or more reference nucleotide sequences each comprising a residue nucleotide sequence of a signature region found in a type II DNA topoisomerase, the signature zone having a defined length which is similar in all species of a chosen group and varies significantly in residue nucleotide sequence among species in the chosen group, the signature region further being flanked by left and right consensus regions having respective residue nucleotide sequences which do not vary significantly among the species;
• obtaining one or more nucleic acid segments from the sample, each said segment derived from the signature region;
• determining a residue nucleotide sequence of at least one of the segments;
• and
• comparing the determined residue nucleotide sequence with said one or more reference nucleotide sequences in the database to find a matching reference nucleotide sequence, and thereby identify the sample organism.

A method of identifying one or more species of organisms in a sample, comprising the steps of:

• providing a database including SEQ. ID Nos. 33-100, the database further containing a plurality of reference sequences each comprising a subunit sequence of a selected signature region of a macromolecule selected from the group consisting of type II topoisomerase enzymes and homologues of type II topoisomerase enzymes;
• each of the reference sequences being specific to a different individual species of a chosen group, and the selected macromolecule further having first and second conserved regions adjacently flanking the selected signature region;
• providing a primer composition comprising at least first and second primers constructed to bind to a DNA encoding the macromolecule in respective first and second DNA flanking regions which respectively encode the first and second conserved regions;
• making an extract of DNA molecules from a sample;
• selectively amplifying desired DNA segments in the extract using the primer composition to produce amplified DNA segments;
• determining one or more nucleotide sequences of individual segments selected from the amplified DNA segments;
• and
• comparing the nucleotide sequences to the database to match the nucleotide sequences with reference sequences of the macromolecule derived from known species to thereby diagnose the presence in the sample of the known species.

A method of identifying one or more species of organisms in a single sample, comprising the steps of:

• extracting DNA from a sample to produce an extract containing a plurality of DNA molecules encoding a type II topoisomerase;
• providing a primer composition comprising at least a pair of DNA primers constructed such that one member of the pair binds respectively to each of two DNA sequences coding respectively for two flanking consensus zones which flank a unique segment of the type II topoisomerase, the unique segment being different among different species and the consensus zones being substantially conserved among different species;
• selectively amplifying a region containing the unique segment of the DNA molecules in the extract using the primer composition to produce a plurality of amplified nucleic acid segments;
• determining one or more DNA sequences corresponding to the amplified unique segments;
• and
• comparing the determined DNA sequences to a database of reference sequences reflective of the variable zone of the topoisomerase II, each of the reference sequences indicative of a different species in a group of organisms, to identify one or more organisms present in the sample, said database including SEQ. ID Nos. 33-100.

A method of identifying one or more species of organisms in a single sample, comprising the steps of:

• extracting DNA from a sample to produce an extract containing a plurality of DNA molecules encoding a type II topoisomerase;
• providing a primer composition comprising at least a pair of DNA primers constructed such that one member of the pair binds respectively to each of two DNA sequences coding respectively for two flanking consensus zones which flank a unique segment of the type II topoisomerase, the unique segment being different among different species and the consensus zones being substantially conserved among different species, wherein the primer composition includes a plurality of primer pairs selected from the group consisting of SEQ. ID Nos. 105 and 106 and sequences differing from SEQ. IDs No. 105 or No. 106 by one or more of the potential substitutions shown in FIG. 3;
• selectively amplifying a region containing the unique segment of the DNA molecules in the extract using the primer composition to produce a plurality of amplified nucleic acid segments;
• determining one or more DNA sequences corresponding to the amplified unique segments;
• and
• comparing the determined DNA sequences to a database of reference sequences reflective of the variable zone of the topoisomerase II, each of the reference sequences indicative of a different species in a group of organisms, to identify one or more organisms present in the sample.

A method of constructing a database of signature sequences of a single protein useful to identify organisms in a sample, comprising:

• a first step of making an extract of DNA from a sample containing DNA from a single known species, and including a DNA molecule coding for a type II DNA topoisomerase;
• a second step of amplifying DNA segments each encoding a selected region of the type II DNA topoisomerase, the selected region comprising a signature region having an amino acid sequence which varies among species in a group, an upstream conserved region and a downstream conserved region, said upstream and downstream conserved regions having amino acid sequences which are substantially conserved among species in a group;
• a third step of determining the individual DNA sequence of at least one of the amplified DNA segments;
• a fourth step of storing data reflective of the determined individual DNA sequence in a database, said database including sequences of region 6 shown in FIG. 2 and sequences of regions 16 and 18 shown in FIGS. 5A and 5B;
• a fifth step of converting the individual DNA sequence to an encoded amino acid sequence;
• and
• a sixth step of repeating said first through fifth steps for a plurality of samples each containing a different single individual species and of storing the encoded amino acid sequence in the database.

A method of constructing a database of signature sequences of a single protein useful to identify organisms in a sample, comprising:

• a first step of making an extract of DNA from a sample containing DNA from a single known species, and including a DNA molecule coding for a type II DNA topoisomerase;
• a second step of amplifying DNA segments each encoding a selected region of the type II DNA topoisomerase, the selected region comprising a signature region having an amino acid sequence which varies among species in a group, an upstream conserved region and a downstream conserved region, said upstream and downstream conserved regions having amino acid sequences which are substantially conserved among species in a group, said step of amplifying DNA segments including the step of providing a DNA primer composition which contains at least one pair of primers having one member of the pair encoding the upstream conserved region and the other member encoding an antisense translation of the downstream conserved region, wherein the primer composition includes primers at least one of the DNA sequences selected from the group consisting of:
• SEQ. ID No. 105, SEQ. ID No. 106, sequences coding for SEQ. ID No. 103, sequences complementary to a sequence coding for SEQ. ID No. 104;
• a third step of determining the individual DNA sequence of at least one of the amplified DNA segments;
• a fourth step of storing data reflective of the determined individual DNA sequence in a database;
• and
• a fifth step of repeating said first through fourth steps for a plurality of samples each containing a different single individual species.