Expedited quantification of mutant ribosomal RNA by binary deoxyribozyme (BiDz) sensors.
RNA
; 21(10): 1834-43, 2015 Oct.
Article
em En
| MEDLINE
| ID: mdl-26289345
ABSTRACT
Mutations in ribosomal RNA (rRNA) have traditionally been detected by the primer extension assay, which is a tedious and multistage procedure. Here, we describe a simple and straightforward fluorescence assay based on binary deoxyribozyme (BiDz) sensors. The assay uses two short DNA oligonucleotides that hybridize specifically to adjacent fragments of rRNA, one of which contains a mutation site. This hybridization results in the formation of a deoxyribozyme catalytic core that produces the fluorescent signal and amplifies it due to multiple rounds of catalytic action. This assay enables us to expedite semi-quantification of mutant rRNA content in cell cultures starting from whole cells, which provides information useful for optimization of culture preparation prior to ribosome isolation. The method requires less than a microliter of a standard Escherichia coli cell culture and decreases analysis time from several days (for primer extension assay) to 1.5 h with hands-on time of â¼10 min. It is sensitive to single-nucleotide mutations. The new assay simplifies the preliminary analysis of RNA samples and cells in molecular biology and cloning experiments and is promising in other applications where fast detection/quantification of specific RNA is required.
Palavras-chave
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
RNA Bacteriano
/
RNA Ribossômico
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Técnicas Biossensoriais
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DNA Catalítico
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Mutação
Idioma:
En
Revista:
RNA
Assunto da revista:
BIOLOGIA MOLECULAR
Ano de publicação:
2015
Tipo de documento:
Article
País de afiliação:
Estados Unidos