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US 8,574,839 B2Granted
Species-specific primer sets and identification of species-specific DNA sequences using genome fragment enrichment
AssigneeU.S. Environmental Protection Agency
InventorsOrin C. Shanks, Jorge Santo Domingo, James E. Graham, Jingrang Lu
Life of this patent
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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, particularly for identifying sources of fecal pollution.
Claims13
A method for identifying a source of fecal contamination in water which comprises
- (a) providing a DNA sample consisting essentially of genomic DNA fragments of microbial community DNA isolated from a sample of fecal contamination isolated from water,
- (b) amplifying genomic DNA in that DNA sample that is specific to a first possible source of said fecal contamination, by means of at least one pair of PCR primers specific to said first possible source, or at least one first source-specific combination of pairs of PCR primers, to obtain a first source-specific PCR amplification product, and
- (c) detecting said first source-specific PCR amplification product, wherein said first source-specific primer pairs or combination of pairs was identified by genomic fragment enrichment (GFE), wherein identifying by GFE comprises:
- (1) providing labeled sheared total genomic DNA from a first microbial species,
- (2) providing PCR amplification-blocked sheared total genomic DNA from a second and different microbial species,
- (3) prehybridizing said labeled DNA of (1) with said blocked DNA of (2) to obtain prehybridized DNA,
- (4) self hybridizing said prehybridized DNA of (1) with PCR-amplified genomic DNA fragments from said first microbial species that comprise or were linked prior to amplification with defined terminal sequence tags, and
- (5) isolating the DNA hybrids resulting from (4), whereby a source of fecal contamination is identified if said first source-specific, PCR amplification product is detected.
5 more elements
A method for identifying a source of fecal contamination in water which comprises
- (a) providing a DNA sample consisting essentially of genomic DNA fragments of microbial community DNA isolated from a sample of fecal contamination isolated from water,
- (b) amplifying any genomic DNA in that DNA sample that is specific to a first possible source of said fecal contamination, by means of at least one pair of PCR primers specific to said first possible source, or at least one first source-specific combination of pairs of PCR primers, to obtain a first source-specific PCR amplification product, and
- (c) detecting said first source-specific PCR amplification product, wherein the first possible source of fecal contamination is chicken, and the primers are chicken-specific,
- wherein at least one pair of PCR primers amplified genomic DNA that is also amplified by a pair of PCR primers selected from the group consisting of the primer pairs CB-R2-42, CB-R2-80, CP-1-10, CP1-24, CP1-26, CP1-40, CP1-55, CP1-74, CP2-9, CP2-57, CP3-46, and CP3-49
- whereby a source of fecal contamination in water is identified if said first source-specific, PCR amplification product is detected.
2 more elements
A method of genome fragment enrichment (GFE) which comprises
- (a) providing labeled sheared total genomic DNA from a first microbial species,
- (b) providing PCR amplification-blocked sheared total genomic DNA from a second and different microbial species,
- (c) prehybridizing said labeled DNA of (a) with said blocked DNA of (b) to obtain prehybridized DNA,
- (d) self hybridizing said prehybridized DNA of (a) with PCR-amplified genomic DNA fragments from said first microbial species that comprise or were linked prior to amplification with defined terminal sequence tags, and
- (e) isolating the DNA hybrids resulting from (d).
2 more elements
Dependencies are inferred from the claim text.