Restriction site tagged (RST) microarrays: a novel technique to study the species composition of complex microbial systems.
We have developed a new type of microarray, restriction site tagged (RST), for example NotI, microarrays. In this approach only sequences surrounding specific restriction sites (i.e. NotI linking clones) were used for generating microarrays. DNA was labeled using a new procedure, NotI representation, where only sequences surrounding NotI sites were labeled. Due to these modifications, the sensitivity of RST microarrays increases several hundred-fold compared to that of ordinary genomic microarrays. In a pilot experiment we have produced NotI microarrays from Gram-positive and Gram-negative bacteria and have shown that even closely related Escherichia coli strains can be easily discriminated using this technique. For example, two E.coli strains, K12 and R2, differ by less than 0.1% in their 16S rRNA sequences and thus the 16S rRNA sequence would not easily discriminate between these strains. However, these strains showed distinctly different hybridization patterns with NotI microarrays. The same technique can be adapted to other restriction enzymes as well. This type of microarray opens the possibility not only for studies of the normal flora of the gut but also for any problem where quantitative and qualitative analysis of microbial (or large viral) genomes is needed.
Zabarovsky
Eugene R
ER
Microbiology and Tumor Biology Center, Department of Cell and Molecular Biology, Karolinska Institute, 171 77 Stockholm, Sweden. eugzab@ki.se
Petrenko
Lev
L
Protopopov
Alexei
A
Vorontsova
Olga
O
Kutsenko
Alexey S
AS
Zhao
Yanyan
Y
Kilosanidze
Gelena
G
Zabarovska
Veronika
V
Rakhmanaliev
Elian
E
Pettersson
Bertil
B
Kashuba
Vladimir I
VI
Ljungqvist
Olle
O
Norin
Elisabeth
E
Midtvedt
Tore
T
Möllby
Roland
R
Winberg
Gösta
G
Ernberg
Ingemar
I
D001419:Bacteria / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism*; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D017422:Sequence Analysis, DNA; D013045:Species Specificity
D004269:DNA, Bacterial; D015252:Deoxyribonucleases, Type II Site-Specific; C038565:GCGGCCGC-specific type II deoxyribonucleases