Open AccessImproved methods for in situ enzymatic amplification and detection of low copy number genes in bacteria
Author(s) -
Jacobs Daniel,
Angles Mark L,
Goodman Amanda E,
Neilan Brett A
Publication year - 1997
Publication title -
fems microbiology letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.899
H-Index - 151
eISSN - 1574-6968
pISSN - 0378-1097
DOI - 10.1111/j.1574-6968.1997.tb10410.x
Subject(s) - in situ , plasmid , nucleic acid , biology , low copy number , bacteria , escherichia coli , gene , microbiology and biotechnology , nucleotide , biochemistry , chemistry , genetics , organic chemistry
We present alternative and improved protocols for in situ analysis of single copy genes in prokaryotes. Primed in situ amplification (PRINS) and cycle PRINS were used to detect, via the incorporation of a fluorescein labelled nucleotide, the presence of specific genes carried on both high and low copy number plasmids in individual cells of Escherichia coli and a marine bacterium, SW5. The optimised protocols described enabled a significant reduction in non‐specific signals whilst maintaining high fluorescent activity via labelled nucleotide incorporation. In addition, nucleic acids were amplified linearly and were retained within the permeabilised microbial cells. These methods provide considerable advances in sensitivity, specificity and reliability compared to current protocols for bacterial in situ nucleic acid amplification.