Single-Point Genome Signature Tags

Abstract

Disclosed is a method for analyzing the organismic complexity of a sample through analysis of the nucleic acid in the sample. In the disclosed method, through a series of steps, including digestion with a type II restriction enzyme, ligation of capture adapters and linkers and digestion with a type IIS restriction enzyme, genome signature tags are produced. The sequences of a statistically significant number of the signature tags are determined and the sequences are used to identify and quantify the organisms in the sample. Various embodiments of the invention described herein include methods for using single point genome signature tags to analyze the related families present in a sample, methods for analyzing sequences associated with hyper- and hypo-methylated CpG islands, methods for visualizing organismic complexity change in a sampling location over time and methods for generating the genome signature tag profile of a sample of fragmented DNA.

1 .- 49 . (canceled) 
     
     
         50 . A method for analyzing the variety of members of specific phyla or families of organisms contained in a sample using single point genome signature tags comprising the steps of:
 a) providing a sample containing one or more organisms;   b) isolating the DNA from the organisms in the sample;   c) contacting the DNA with a fragmenting enzyme under conditions appropriate for substantially complete digestion of the DNA thereby generating a plurality of DNA fragments, each having complementary cohesive termini, said fragmenting enzyme being a type II restriction endonuclease which does not cleave within conserved segments of a gene of focus, said gene of focus being a gene containing segments that are highly conserved across a phylum or a family of organisms and segments that are species-specific across the phylum or family of organisms;   d) incubating the DNA fragments of step c) with a molar excess of a duplex linker having a type IIS restriction enzyme recognition sequence and one cohesive terminus compatible with termini generated by the fragmenting enzyme of step c), under conditions appropriate for ligating one duplex linker to each cohesive terminus of the DNA fragments thereby generating a plurality of DNA fragment-duplex linker species;   e) amplifying a portion of a specific subset of DNA fragment-duplex linker species using a pair of primers comprising a first primer specific for the duplex linker and an anchoring primer, said anchoring primer being specific for a conserved segment of the gene of focus and which anchoring primer is covalently modified with a first member of a specific binding pair, thereby generating a mixture of unamplified DNA fragment-duplex linker species and amplified portions of a subset of the DNA fragment-duplex linker species, said amplified portions comprising sequences that are conserved across the phylum or family and sequences that are species-specific and which species-specific sequences contain the single point genome signature tags;   f) capturing the amplified portions of the subset by contacting the mixture of step e) with a solid support having an attached second member of the specific binding pair;   g) incubating the solid support and captured amplified portions of step f) with the type IIS restriction enzyme, under conditions appropriate for substantially complete digestion thereby releasing the duplex linkers, each having an appended single point genome signature tag (SP-GST);   h) recovering the released duplex linkers and appended SP-GSTs;   i) incubating the recovered linkers and SP-GSTs of step h) with a molar excess of an amplification adapter, the amplification adapter having one terminus compatible with the termini of the appended SP-GSTs, the incubation being carried out under conditions appropriate for ligating one amplification adapter to each appended SP-GST;   j) recovering the ligation product of step i);   k) determining the nucleotide sequence of a statistically significant number of appended SP-GSTs to generate a listing of SP-GSTs; and,   l) relating the listing of SP-GSTs of step k) to DNA sequences in databases to analyze the variety of members of specific phyla or families of organisms contained in the sample.   
     
     
         51 . The method of  claim 50  in which the SP-GSTs are located upstream or downstream of the gene of focus. 
     
     
         52 . The method of  claim 50  in which the SP-GSTs are located within the gene of focus. 
     
     
         53 . The method of  claim 50  wherein the gene of focus is selected from the group consisting of rDNA genes of archaebacteria, rDNA genes of eubacteria, rDNA genes of eukaryotes, rDNA genes of fungi or rDNA genes of organelles. 
     
     
         54 . The method of  claim 50  wherein the gene of focus is a gene or locus that is conserved among related organisms. 
     
     
         55 . The method of  claim 50  wherein the gene of focus is a gene or locus encoding an enzyme of a pathway that is conserved among related organisms. 
     
     
         56 .- 94 . (canceled) 
     
     
         95 . The method according to  claim 50  wherein
 the specific binding pair is selected from the group consisting of biotin/streptavidin, antigen/antibody, sugar/lectin, apoenzyme/cofactor, hormone/receptor, enzyme/inhibitor, and complementary homopolymeric oligonucleotides;   the type IIS restriction enzyme is MmeI;   the solid support is selected from the group consisting of magnetic beads, glass beads, filter membranes, filter papers and polymeric beads; and,   the nucleotide sequence is determined by either capillary gel electrophoresis or pyrosequencing.   
     
     
         96 . The method according to  claim 95  wherein the gene of focus is selected from the group consisting of rDNA genes of archaebacteria, rDNA genes of eubacteria, rDNA genes of eukaryotes, rDNA genes of fungi or rDNA genes of organelles. 
     
     
         97 . The method according to  claim 50  wherein the amplification adapter is part of a collection comprising amplification adapters with degenerate overhang sequences. 
     
     
         98 . The method according to  claim 95  wherein the amplification adapter is part of a collection comprising amplification adapters with a subset of degenerate overhang sequences.