Back to results
US 2012/0058485 A1Published application
Rapid and comprehensive identification of prokaryotic organisms (continuation)
OwnerPeter Kaith Roganinventor
Life of this patent
Estimating the term…
Abstract
An improved method for rapid identification of microorganisms is disclosed, along with sequences of PCR primers optimized for this purpose. The primers are designed based on information analysis of sequences from a large number of organism to amplify certain segments of genomic DNA whose sequences are unique among different organisms. The PCR products are compared with a DNA sequence database to obtain the identity of the microorganisms. This approach provides an accurate and fast identification and taxonomic assignment of microbial species.
Claims15
A method for obtaining data for taxonomic assignment of unknown species comprising:
- (a) selecting from more than one known species a divergent segment of DNA with low average information content surrounded by two conserved segments of DNA wherein said conserved segments comprise DNA segments with high average information content and wherein information content is determined by average information in bits of a related set of sequences and represents the total sequence conservation calculated by:
- Rsequence2=−[−Σf(b,l) log2f(b,l)+e(n(l))]
- b=[A,G,C,T]
- wherein
- f(b,l) is the frequency of each base b (A,G,C, and T) at position l, and
- e(n1) is a generalized correction term determined by Shannon's Uncertainty for a sample size n at position l,
- (b) selecting primers for PCR amplification of said divergent segment such that said primers anneal to said conserved segments in any of these one or more unknown species, wherein each primer contains a mixture of nucleotides in which each nucleotide is present at the same proportion as it is present in the set of sequences of the known species used to compute the information content;
- (c) amplifying said divergent segment of DNA by PCR technique using said primers to obtain PCR products;
- (d) separating said PCR products;
- (e) comparing said separated PCR products with a database consisting of properties that can be converted or derived from a subject sequence and from similar sequences distinguishable from the subject sequence, wherein said properties are selected from the group consisting of nucleotide composition, base composition, nucleotide sequence, DNA structure, mass ratio of the DNA molecules or fragments derived therefrom, chemical reactivity of the DNA molecules, binding properties to other DNA molecules or proteins, thermal stability, and combination thereof;
- (f) measuring a taxonomic distance of said properties between said separated PCR products and organisms in said database;
- and
- (g) assigning a taxonomic reference to said separated PCR products based upon said properties, wherein the PCR products are separated by means selected from the group consisting of magnetic separation, gel purification, mass spectrometry and combination thereof, by limiting dilution of mixtures of amplicons into multiple aliquots, each aliquot containing a distinct individual DNA molecule.
10 more elements
A method for identifying an organism in a sample, comprising:
- (a) selecting from more than one known species a divergent segment of DNA with low average information content surrounded by two conserved segments of DNA wherein said conserved segments comprise DNA segments with high average information content and wherein information content is determined by average information in bits of a related set of sequences and represents the total sequence conservation calculated by:
- Rsequence2=−[−Σf(b,l) log2 f(b,l)+e(n(l))]
- b==[A,G,C,T]
- wherein
- f(b,l) is the frequency of each base b (A,G,C, and T) at position l, and
- e(n(l) is a generalized correction term determined by Shannon's Uncertainty for a sample size n at position l,
- (b) selecting primers for PCR amplification of said divergent segment such that said primers anneal to said conserved segments in any of these one or more unknown species, wherein each primer contains a mixture of nucleotides in which each nucleotide is present at the same proportion as it is present in the set of sequences of the known species used to compute the information content;
- (c) amplifying said divergent segment of DNA by PCR technique using said primers to obtain PCR products;
- (d) separating said PCR products;
- (e) comparing said separated PCR products with a database consisting of properties that can be converted or derived from a subject sequence and from similar sequences distinguishable from the subject sequence, wherein said properties are selected from the group consisting of nucleotide composition, base composition, nucleotide sequence, DNA structure, mass ratio of the DNA molecules or fragments derived therefrom, chemical reactivity of the DNA molecules, binding properties to other DNA molecules or proteins, thermal stability, and combination thereof;
- and
- (f) identifying one or more organisms in said separated PCR products based upon properties, said separation step comprising limiting dilution of mixtures of amplicons into one or more aliquots, each aliquot containing either a distinct single DNA molecule or a homogenous group of DNA molecules having the same sequence.
9 more elements
A method for identifying an organism in a sample, comprising:
- (a) selecting from more than one known species a divergent segment of DNA with low average information content surrounded by two conserved segments of DNA wherein said conserved segments comprise DNA segments with high average information content and wherein information content is determined by average information in bits of a related set of sequences and represents the total sequence conservation calculated by:
- Rsequence=2−[−Σf(b,l) log2 f(b,l)+e(n(l))]
- b==[A,G,C,T]
- wherein
- f(b,l) is the frequency of each base b (A,G,C, and T) at position l, and
- e(n(1) is a generalized correction term determined by Shannon's Uncertainty for a sample size n at position l,
- (b) selecting primers for PCR amplification of said divergent segment such that said primers anneal to said conserved segments in any of these one or more unknown species, wherein each primer contains a mixture of nucleotides in which each nucleotide is present at the same proportion as it is present in the set of sequences of the known species used to compute the information content;
- (c) amplifying said divergent segment of DNA by PCR technique using said primers to obtain PCR products;
- (d) separating said PCR products;
- (e) comparing said separated PCR products with a database consisting of properties that can be converted or derived from a subject sequence and from similar sequences distinguishable from the subject sequence, wherein said properties are selected from the group consisting of nucleotide composition, base composition, nucleotide sequence, DNA structure, mass ratio of the DNA molecules or fragments derived therefrom, chemical reactivity of the DNA molecules, binding properties to other DNA molecules or proteins, thermal stability, and combination thereof;
- and
- (f) identifying one or more organisms in said separated PCR products based upon properties, wherein said divergent segment of DNA surrounded by said conserved segments is performed in the region of the DNA coding for the ribosomal RNA of any organism, said ribosomal RNA is the 28S ribosomal RNA from eukaryotic organisms or the 16S ribosomal RNA from prokaryotic organisms, and the separated PCR products are characterized identified based upon nucleotide sequence.
9 more elements
Dependencies are inferred from the claim text.