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

• US 5,645,994 A
• Filed: 06/06/1995
• Issued: 07/08/1997
• Est. Priority Date: 07/05/1990
• Status: Expired due to Term

• Current Assignee: University of Utah Research Foundation (State of Utah)
• Original Assignee: University of Utah Research Foundation (State of Utah)
• Inventors: Huang, Wai Mun
• Primary Examiner(s): Marschel, Ardin H.
• Application Number:
  US08/470,179
• Time in Patent Office: 763 Days
• Field of Search: 435/5, 435/6, 435/91.3, 435/810, 436/501, 536/22.1, 536/23.1, 536/24.1, 536/24.3-24.33, 935/77, 935/78
• US Class Current: 435/6.14
• CPC Class Codes: C12N 15/10 Processes for the isolation, preparation or purification of DNA or RNA chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34
• CPC Class Codes: C12N 9/88 Lyases (4.)
• CPC Class Codes: C12Q 1/689 for bacteria
• CPC Class Codes: C12Q 1/6893 for protozoa
• CPC Class Codes: C12Q 1/6895 for plants, fungi or algae
• CPC Class Codes: Y10S 435/81 Packaged device or kit

Abstract

A method of identifying species in a sample is based on pairs of consensus amino acid segments which flank variable amino acid segments of type II DNA topoisomerases. In one embodiment, a DNA primer composition termed "universal primers", is used to amplify the DNA segments coding for the variable or "signature" amino acid sequences. The amplified segments are then cloned and sequenced, and the DNA sequence is matched against a database of signature sequences from multiple species. The signature DNA sequences may be convened to the amino acid "signature" sequences for which they code, and these signature sequences matched against a database of amino acid reference sequences, thereby determining which species were present in the original sample. The universal DNA primers are useful to detect and identify multiple unknown microbial species in a sample. In another embodiment, a specific primer pair specific for a particular organism is provided, which has sequences derived from the signature regions between the flanking conserved regions. A general method for selecting a suitable protein and producing universal primers based upon it is also provided.

Claims

1. A method of selectively amplifying DNA segments of one or more species of organisms in a sample, comprising the steps of:

providing a database 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 universal primer composition comprising a primer pair constructed to bind to a DNA encoding the macromolecule;

making an extract of DNA molecules from a sample; and

using the primer composition to selectively amplify DNA segments of the signature region in the extract to produce amplified DNA segments.

2. The method of claim 1, wherein said universal primer composition is constructed to bind to third and fourth DNA regions which respectively encode third and fourth portions of the signature region, the third and fourth portions being located between the first and second conserved regions.

3. The method of claim 2, further including the steps of:

providing a consensus primer composition comprising primers constructed to bind to first and second DNA flanking regions which respectively encode the first and second conserved regions; and

amplifying the extract with the consensus primer composition prior to said step of amplifying with the primer pair.

4. The method of claim 2, wherein the primer pair has sequences selected to amplify a region selected from the group consisting of:

region 810 in FIG. 8; and

region 910 in FIGS. 9A-9B.

5. The method of claim 4, wherein the specific primer composition is selected from the group consisting of the following primer pairs:

SEQ ID Nos. 196-197, SEQ ID Nos. 198-199, SEQ ID Nos. 200-201, SEQ ID Nos. 202-203, SEQ ID Nos. 204-205, and SEQ ID Nos. 206-207; and

wherein the specific primer composition may further include additional primer pairs changed from the selected primer pair by a nucleotide substitution which does not change the coded residue sequence.

6. The method of claim 2, wherein the primer pair is constructed to amplify a segment encompassed within a sequence selected from the group consisting of:

SEQ IDs Nos. 33-100 and 107-195.

7. The method of claim 2, wherein the universal primer composition includes a combination of two or more specific primer pairs derived from the signature regions of different species.

8. The method of claim 2, wherein said step of providing the database includes 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).

9. The method of claim 2, wherein the group may include any of the following species:

bacterial species, plant species, fungal species, and protozoan species.

10. The method of claim 2, wherein the database includes SEQ ID Nos. 1-100 and 107-195.

11. The method of claim 3, wherein said step of providing a consensus primer composition comprises providing a plurality of DNA primers having different sequences which are selected to operably bind to alternate DNA segments coding for the conserved regions flanking the subunit sequence.

12. The method of claim 3, further including the steps of:

determining a residue sequence of at least one individual segment selected from the DNA segments amplified with the first and second primers; and

comparing the residue sequence with a reference signature sequence of the macromolecule derived from a known species to thereby diagnose the presence in the sample of the known species.

13. The method of claim 12, wherein said steps of determining a residue sequence and comparing the residue sequence are performed for each of a plurality of individual DNA segments, thereby identifying a plurality of organisms present in the sample.

14. The method of claim 12, wherein the residue sequence and the reference sequences are nucleotide sequences.

15. The method of claim 12, further including a step of converting the determined residue sequence to an amino acid sequence and wherein the database further includes reference amino acid sequences.

16. 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.

17. The method of claim 16, which further includes the steps of:

providing a consensus 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; and

subjecting said extract to amplification using the consensus primer composition to produce a plurality of amplified signature segments each spanning the signature region, said step of subjecting to consensus amplification being performed prior to said step of amplification with the specific primer composition.

18. The method of claim 16, wherein the specific primer composition has a sequence coding for regions selected from the group consisting of:

regions 806 and shown in FIG. 8; and

regions 906 and 908 in FIGS. 9A-9B.

19. The method of claim 16, wherein the specific primer composition is selected from the group consisting of the following primer pairs:

SEQ ID Nos. 196-197, SEQ ID Nos. 198-199, SEQ ID Nos. 200-201, SEQ ID Nos. 202-203, SEQ ID Nos. 204-205, and SEQ ID Nos. 206-207; and

wherein the specific primer composition may include additional primer pairs changed from the selected primer pair by a nucleotide substitution which does not change the coded residue sequence.

20. The method of claim 16, further including a step of providing additional specific primer compositions, wherein the amplification step is performed using a combination of two or more specific primer compositions specific for different organisms.

21. The method of claim 16, wherein the organism is selected from the group consisting of:

bacterial species, plant species, fungal species, and protozoan species.

22. The method of claim 16, wherein the organism is a bacterial species.

23. The method of claim 16, wherein the specific primer composition is constructed to amplify a segment encompassed within a sequence selected from the group consisting of:

SEQ IDs Nos. 1-100 and 107-195.

24. The method of claim 17, wherein the consensus primer composition comprises a plurality of primer pairs selected from SEQ ID Nos. 105 and 106 and sequences substantially similar to these according to FIG. 3.

25. The method of claim 17, which further includes a step of providing a database of reference sequences, wherein the reference sequences include sequences selected from the group consisting of SEQ ID Nos. 1-100 and 107-195.

26. 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.

27. The method of claim 26, wherein the sample organism is selected from the group consisting of plant species, fungal species and protozoan species.

28. 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.

29. 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.

30. The method of claim 29, wherein said steps of determining nucleotide sequences and comparing the nucleotide sequences are performed for a plurality of the amplified DNA segments.

31. The method of claim 29, wherein the subunit sequence is an amino acid sequence, and said step of comparing the nucleotide sequences further includes decoding the nucleotide sequences to signature amino acid sequences and comparing the signature amino acid sequences to the reference sequences.

32. The method of claim 29, wherein said steps of determining a nucleotide sequence and comparing the nucleotide sequence are performed for each of a plurality of individual the DNA segments, thereby identifying a plurality of organisms present in the sample.

33. The method of claim 29, wherein the macromolecule is further selected to have an internal conserved segment embedded within the signature sequence.

34. 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.

35. The method of claim 34, wherein said steps of determining DNA sequences and comparing said determined sequences are performed for a plurality of the amplified nucleic acid segments.

36. The method of claim 35, wherein said step of selectively amplifying comprises the steps of:

providing at least one pair of primers useful to amplify the selected DNA segment by the technique of polymerase chain reaction; and

incubating the primers with the DNA extract and other necessary reagents for synthesis of DNA molecules under PCR amplification conditions to produce the selectively amplified segments.

37. 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.

38. 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.

39. The method of claim 38, wherein the species are bacterial species.

40. The method of claim 38, wherein the species are selected from the group consisting of fungal species, plant species and protozoan species.

41. 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.

42. A composition of matter comprising at least two single-stranded DNA primers capable of selective hybridization to amplify an intervening signature segment of a type II topoisomerase, said signature segment being selected from the group consisting of segment 6 of FIG. 2, and segments 16 and 18 of FIGS. 5A and 5B.

43. The composition of claim 42, which includes primers having 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, and sequences complementary to a sequence coding for SEQ. ID No. 104.