Method of identifying putative antibiotic resistance genes
AssigneeUniversity of Rochester
InventorsBarry G. Hall, Miriam Barlow
Life of this patent
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Abstract
The present invention relates to methods of identifying putative antibiotic resistance genes. According to one embodiment, this is carried out by first isolating a microbial DNA molecule or cDNA which encodes a putative antibiotic resistance protein or polypeptide and then determining whether the microbial DNA molecule or cDNA confers resistance against an antibiotic agent. According to another embodiment, this is carried out by first determining whether a microbial DNA molecule or cDNA confers resistance against an antibiotic agent when the microbial DNA molecule is expressed in its native cell following transformation of the cell and then isolating the microbial DNA molecule or cDNA which confers resistance against the antibiotic agent.
Claims28
A method of identifying putative antibiotic resistance genes comprising:
- isolating a microbial DNA molecule or cDNA which encodes a putative antibiotic resistance protein or polypeptide that does not confer an antibiotic resistance phenotype to the microbe from which the DNA or cDNA was isolated;
- transforming a host cell with the microbial DNA molecule or cDNA under conditions effective to express the putative antibiotic resistance protein or polypeptide;
- and
- growing transformed host cells on a growth medium including the antibiotic agent, wherein proliferation of host cells indicates antibiotic resistance conferred by expression of the putative antibiotic resistance protein or polypeptide.
A method of identifying putative antibiotic resistance genes comprising:
- determining whether a microbial DNA molecule confers resistance against an antibiotic agent when the microbial DNA molecule is expressed in its native cell following transformation of said cell with a transposon comprising, in a single DNA molecule, a pair of inverted repeats at opposite ends of the single DNA molecule and a promoter region oriented in a manner that affords transcription of coding regions located outside of the transposon sequence following its insertion into the genomic DNA of said cell, and but for transformation of said cell the microbial DNA molecule does not confer antibiotic resistance to the native cell, and
- isolating the microbial DNA molecule or cDNA which confers resistance against the antibiotic agent.
A method of identifying putative antibiotic resistance genes comprising, in combination:
- (A) identifying a microbial DNA molecule or cDNA from a pool of chromosomal, microbial DNA molecules or their corresponding cDNAs, the microbial DNA molecule or cDNA encoding a putative antibiotic resistance protein or polypeptide, which microbial DNA or cDNA does not confer a resistance phenotype to the mocrobial source thereof,
- isolating the microbial DNA molecule or cDNA, and
- determining whether the microbial DNA molecule or cDNA confers to a first host cell resistance against an antibiotic agent;
- and
- (B) introducing a transposon into genomic DNA of a second host cell of the same species from which the microbial DNA molecule or cDNA was isolated in (A), the transposon comprising, in a single DNA molecule, a pair of inverted repeats at opposite ends of the DNA molecule and a promoter region which is oriented in a manner that affords transcription of coding regions located outside of the transposon sequence following its insertion into the genomic DNA, and
- growing transformed host cells on a selection media comprising an antibiotic agent, wherein proliferation of host cells indicates that the microbial DNA molecule is expressed and confers resistance against the antibiotic agent.
Dependencies are inferred from the claim text.