Laser capture microdissection of bacterial cells targeted by fluorescence in situ hybridization
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Laser capture microdissection of bacterial cells targeted by fluorescence in situ hybridization. / Schou, Kirstine Klitgaard; Mølbak, Lars; Jensen, Tim Kåre; Lindboe, Christian Frederik; Boye, Mette.
In: BioTechniques, Vol. 39, No. 6, 2005.Research output: Contribution to journal › Journal article › Research › peer-review
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TY - JOUR
T1 - Laser capture microdissection of bacterial cells targeted by fluorescence in situ hybridization
AU - Schou, Kirstine Klitgaard
AU - Mølbak, Lars
AU - Jensen, Tim Kåre
AU - Lindboe, Christian Frederik
AU - Boye, Mette
PY - 2005
Y1 - 2005
N2 - Direct cultivation-independent sequence retrieval of unidentified bacteria from histological tissue sections has been limited by the difficulty of selectively isolating specific bacteria from a complex environment. Here, a new DNA isolation approach is presented for prokaryotic cells. By this method, a potentially pathogenic strain of the genus Brachyspira from formalin-fixed human colonic biopsies were visualized by fluorescence in situ hybridization (FISH) with a 16S rRNA-targeting oligonucleotide probe, followed by laser capture microdissection (LCM) of the targeted cells. Direct 16S rRNA gene PCR was performed from the dissected microcolonies, and the subsequent DNA sequence analysis identified the dissected bacterial cells as belonging to the Brachyspira aalborgi cluster 1. The advantage of this technique is the ability to combine the histological recognition of the specific bacteria within the tissue with molecular analysis of 16S rRNA gene or other genes of interest. This method is widely applicable for the identification of noncultivable bacteria and their gene pool from formalin-fixed paraffin-embedded tissue samples.
AB - Direct cultivation-independent sequence retrieval of unidentified bacteria from histological tissue sections has been limited by the difficulty of selectively isolating specific bacteria from a complex environment. Here, a new DNA isolation approach is presented for prokaryotic cells. By this method, a potentially pathogenic strain of the genus Brachyspira from formalin-fixed human colonic biopsies were visualized by fluorescence in situ hybridization (FISH) with a 16S rRNA-targeting oligonucleotide probe, followed by laser capture microdissection (LCM) of the targeted cells. Direct 16S rRNA gene PCR was performed from the dissected microcolonies, and the subsequent DNA sequence analysis identified the dissected bacterial cells as belonging to the Brachyspira aalborgi cluster 1. The advantage of this technique is the ability to combine the histological recognition of the specific bacteria within the tissue with molecular analysis of 16S rRNA gene or other genes of interest. This method is widely applicable for the identification of noncultivable bacteria and their gene pool from formalin-fixed paraffin-embedded tissue samples.
U2 - 10.2144/000112024
DO - 10.2144/000112024
M3 - Journal article
VL - 39
JO - BioTechniques
JF - BioTechniques
SN - 0736-6205
IS - 6
ER -
ID: 339892970