Species-specific primer sets and identification of species-specific DNA sequences using genome fragment enrichment
Inventors
Shanks, Orin C. • Domingo, Jorge Santo • Graham, James E. • Lu, Jingrang
Assignees
United States, As Represented By Us Environmental Protection Agency • US Environmental Protection Agency
Publication Number
US-7572584-B2
Publication Date
2009-08-11
Expiration Date
2025-12-27
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Abstract
Targeted sequencing of genetic regions that differ between two DNA preparations uses genomic fragment enrichment. This method can be used to study genetic variation among closely related species and microbial communities.
Core Innovation
The invention presents a method called Genome Fragment Enrichment (GFE) that uses a specific solution phase competitive DNA hybridization technique to identify species, strain, and host-specific microbial DNA sequences. This method enriches and isolates unique DNA fragments from one microbial community or genome relative to another, enabling the detection of genetic differences. The approach involves labeling genomic DNA fragments from a first microbial community or species, hybridizing these with DNA fragments from a second community or species, and then capturing uniquely hybridized DNA sequences using defined terminal sequence tags and PCR amplification. This allows for the identification of unique DNA sequences useful for differentiating closely related microbial species or microbial communities from different hosts.
The problem addressed by the invention arises from limitations of current microbial source tracking (MST) methods used in environmental water quality monitoring. Existing approaches, such as plate culture methods and PCR strategies targeting 16S rDNA genes, cannot accurately discriminate among closely related bacterial strains or animal sources of fecal contamination. These limitations stem from the inability of current methods to target microorganism DNA sequences encoding proteins directly involved in host-microbe interactions, which are expected to exhibit higher genetic variation. Moreover, whole genome sequencing for comparative microbial analysis remains expensive and impractical for routine use. The invention aims to overcome these deficiencies by providing a practical, positive selection method to identify unique genetic markers specific to microbial species, strains, and host origins.
The invention also addresses the need for precise identification of fecal pollution sources from specific animals to support regulatory water quality standards and management practices, such as those mandated under the U.S. Clean Water Act's Total Maximum Daily Load (TMDL) requirements. By enabling the isolation of species- and host-specific DNA sequences from microbial communities in fecal material, the method facilitates the development of PCR primer sets for accurate detection and differentiation of pollution origins. The technique's successful application to differentiate between Enterococcus species and to identify cow-, chicken-, and human-specific DNA sequences demonstrates its broad utility in microbial ecology, pathogen detection, and environmental monitoring.
Claims Coverage
The patent contains two independent claims directed to methods for identifying genetic differences between microbial communities or genomes, with multiple dependent claims specifying particular embodiments.
Method for identifying differences between microbial communities
A method comprising obtaining labeled first genomic DNA fragments from a first microbial community and hybridizing them with genomic DNA fragments from a second community; incubating these with additional genomic fragments from the first community containing defined terminal sequence tags to form DNA hybrids; capturing the formed DNA hybrids using the tags; performing PCR amplification selectively on the tagged fragments; obtaining enriched DNA sequences unique to the first community; and identifying these enriched sequences to determine differences between the communities.
Method for identifying genetic differences between two microbial genomes
A method comprising obtaining labeled first genomic DNA fragments from a first microorganism and hybridizing them with genomic DNA fragments from a second microorganism; incubating with additional genomic fragments from the first microorganism containing defined terminal sequence tags; capturing resulting DNA hybrids formed with the tags; selectively amplifying tagged fragments by PCR; obtaining enriched sequences unique to the first microorganism; and identifying these sequences to determine genetic differences between the two microbial genomes.
The claims cover methods employing a positive selection Genome Fragment Enrichment process to isolate and identify species- or community-specific DNA sequences by competitive hybridization, labeling, capture, and PCR amplification steps, applicable to microbial communities or genomes including specific microbial species such as Enterococcus.
Stated Advantages
The invention accelerates identification of DNA sequences unique to one microorganism or microbial community relative to another, enabling precise microbial source tracking.
It provides a positive selection method less prone to false positives compared to existing negative selection approaches like SSH or subtractive hybridization.
The method facilitates development of species- and host-specific PCR primers and probes for environmental monitoring, diagnosis, and microbial ecology studies.
The invention allows discrimination among closely related bacterial species, strains, and source origins, overcoming limitations of 16S rDNA-based PCR methods.
Documented Applications
Identifying microbial DNA sequences to determine sources of fecal contamination in environmental waters associated with animals such as cattle, humans, and chickens.
Comparing closely related bacterial genomes, exemplified by Enterococcus faecalis and Enterococcus faecium, to isolate species-specific genetic markers.
Developing PCR primer sets for species- and host-specific microbial source tracking using environmental, fecal, and microbial community DNA samples.
Detecting and quantifying fecal pollution origins for regulatory applications, such as Total Maximum Daily Loads (TMDL) under the Clean Water Act.
Applications in molecular methods to study microbial ecology, clinically relevant species discrimination, environmental monitoring of water quality, and discovery of novel virulence factors.
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