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Method and compositions for identification of species in a sample using type II topoisomerase sequences
| Details |
Inventors: Huang, Wai Mun;
Assignee: University of Utah Research Foundation (Salt Lake City, UT)
Primary Examiner: Marschel; Ardin H.
Assistant Examiner:
Attorney, Agent or Firm: Trask, Britt & Rossa
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. |
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DETAILED DESCRIPTION The invention comprises means to identify one or more species in a sample by selectively amplifying a nucleic acid segment encoding a signature region of a macromolecule which is present in all the organisms of interest.
The macromolecule is selected as having a signature region unique to one or a few species flanked by consensus regions which are substantially conserved among the organisms of interest.
In a highly preferred embodiment, the macromolecule is a type II topoisomerase (also referred to as "gyrase" in bacterial species).
The means for identification comprise a primer composition, together with a method of using the primer composition, optionally in conjunction with a database of residue sequences of the molecule.
The primer composition for the selective amplification may desirably be derived by analysis of a database of residue sequences of the macromolecule.
In one embodiment, the invention includes a universal primer composition comprising primers whose DNA sequences are derived from the amino acid sequence of a type II topoisomerase.
Type II topoisomerase has been found to contain at least one pair of highly conserved consensus amino acid sequences flanking a variable amino acid "signature" segment.
The consensus sequences are selected as being common to the protein from many or all different species, while each signature segment is specific to one or a few species.
The primer pair(s) of the universal primer composition are constructed to hybridize to DNA coding for the consensus sequences.
In a highly preferred embodiment, the universal primer composition comprises a mixture of most or all of the alternate DNA sequences coding for the same amino acid sequence.
A method of using the universal primer composition comprises selectively amplifying from a sample, the nucleic acid segments coding for the signature segment of organisms in the sample, and sequencing a representative sample of the individual amplified segments.
The sequences of individual amplified segments are then each compared to a database containing a plurality of reference signature sequences each corresponding to a particular species
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