Unique marker finder algorithm generates molecular diagnostic markers

By taking advantage of the power of comparative genomics, we devised an algorithm, Unique Marker Finder (U-MarFin), to generate a collection of unique DNA sequences from a target organism. The whole target genome is partitioned into a scoring pool of less 4000 base-pair fragments, which are then subjected to elimination of homologous sequences in other bacterial genomes by BLAST alignment, and looked for all open reading frames as they may be applied as unique markers. Through regular, nested, multiplex and real time PCR and microarray technology, we empirically demonstrated that the sequences discovered were highly specific to the species that they are derived from, and they can serve as molecular biomarkers for diagnostic purpose.

[1]  S. Ratnam,et al.  Latex agglutination test for detection of Escherichia coli serotype O157 , 1989, Journal of clinical microbiology.

[2]  Thomas L. Madden,et al.  Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. , 1997, Nucleic acids research.

[3]  G. Fox,et al.  How close is close: 16S rRNA sequence identity may not be sufficient to guarantee species identity. , 1992, International journal of systematic bacteriology.

[4]  A. Rasooly,et al.  Microarray Analysis of Microbial Virulence Factors , 2001, Applied and Environmental Microbiology.

[5]  L. Fox,et al.  Sensitivity of bacteriologic culture for detection of Escherichia coli O157:H7 in bovine feces , 1995, Journal of clinical microbiology.

[6]  H. Blöcker,et al.  Detection and identification of mycobacteria by amplification of rRNA , 1990, Journal of clinical microbiology.

[7]  N. W. Davis,et al.  Genome sequence of enterohaemorrhagic Escherichia coli O157:H7 , 2001, Nature.

[8]  S. Salzberg,et al.  Alignment of whole genomes. , 1999, Nucleic acids research.

[9]  H. Smith,et al.  ACP Broadsheet 135: January 1993. Isolation and identification methods for Escherichia coli O157 and other Vero cytotoxin producing strains. , 1993, Journal of clinical pathology.

[10]  S. Salzberg,et al.  Versatile and open software for comparing large genomes , 2004, Genome Biology.

[11]  F. Brockman,et al.  Direct Detection of 16S rRNA in Soil Extracts by Using Oligonucleotide Microarrays , 2001, Applied and Environmental Microbiology.

[12]  S. Cole,et al.  The evolution of mycobacterial pathogenicity: clues from comparative genomics. , 2001, Trends in microbiology.

[13]  A. Givaudan,et al.  Plastic architecture of bacterial genome revealed by comparative genomics of Photorhabdus variants , 2008, Genome Biology.

[14]  C. Woodley,et al.  The mtp40 gene is not present in all strains of Mycobacterium tuberculosis , 1996, Journal of clinical microbiology.

[15]  S F Altschul,et al.  Local alignment statistics. , 1996, Methods in enzymology.

[16]  T. Imaeda.  Deoxyribonucleic Acid Relatedness Among Selected Strains of Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium bovis BCG, Mycobacterium microti, and Mycobacterium africanum , 1985 .

[17]  G. Mahairas,et al.  Molecular analysis of genetic differences between Mycobacterium bovis BCG and virulent M. bovis , 1996, Journal of bacteriology.

[18]  J. Guesdon,et al.  Characterization of a Mycobacterium tuberculosis insertion sequence, IS6110, and its application in diagnosis , 1990, Journal of clinical microbiology.

[19]  M. Patarroyo,et al.  Amplification of a species-specific DNA fragment of Mycobacterium tuberculosis and its possible use in diagnosis , 1991, Journal of clinical microbiology.

[20]  W. Pearson.  Empirical statistical estimates for sequence similarity searches. , 1998, Journal of molecular biology.

[21]  N. Morrison,et al.  IS6110 Transposition and Evolutionary Scenario of the Direct Repeat Locus in a Group of Closely Related Mycobacterium tuberculosis Strains , 1998, Journal of bacteriology.

[22]  S T Cole,et al.  Comparative genomics of the mycobacteria. , 2000, International journal of medical microbiology : IJMM.

[23]  D van Soolingen,et al.  Comparison of various repetitive DNA elements as genetic markers for strain differentiation and epidemiology of Mycobacterium tuberculosis , 1993, Journal of clinical microbiology.

[24]  E. Liébana,et al.  Assessment of genetic markers for species differentiation within the Mycobacterium tuberculosis complex , 1996, Journal of clinical microbiology.

[25]  T. Gingeras,et al.  Comparing genomes within the species Mycobacterium tuberculosis. , 2001, Genome research.

[26]  S. Altschul,et al.  Issues in searching molecular sequence databases , 1994, Nature Genetics.

[27]  Gary L. Andersen,et al.  High-Density Microarray of Small-Subunit Ribosomal DNA Probes , 2002, Applied and Environmental Microbiology.

[28]  B. Ross,et al.  Characterization of Mycobacterium tuberculosis strains from Vietnamese patients by Southern blot hybridization , 1993, Journal of clinical microbiology.

[29]  C R Woese,et al.  The phylogeny of prokaryotes. , 1980, Microbiological sciences.

[30]  E. Myers,et al.  Basic local alignment search tool. , 1990, Journal of molecular biology.

[31]  T. Cheasty,et al.  The serological relationship between Yersinia enterocolitica O9 and Escherichia coli O157 using sera from patients with yersiniosis and haemolytic uraemic syndrome , 1991, Epidemiology and Infection.

[32]  S. Altschul.  A protein alignment scoring system sensitive at all evolutionary distances , 1993, Journal of Molecular Evolution.