Influence of dangling ends and surface-proximal tails of targets on probe-target duplex formation in 16S rRNA gene-based diagnostic arrays.
Dangling ends and surface-proximal tails of gene targets influence probe-target duplex formation and affect the signal intensity of probes on diagnostic microarrays. This phenomenon was evaluated using an oligonucleotide microarray containing 18-mer probes corresponding to the 16S rRNA genes of 10 waterborne pathogens and a number of synthetic and PCR-amplified gene targets. Signal intensities for Klenow/random primer-labeled 16S rRNA gene targets were dissimilar from those for 45-mer synthetic targets for nearly 73% of the probes tested. Klenow/random primer-labeled targets resulted in an interaction with a complex mixture of 16S rRNA genes (used as the background) 3.7 times higher than the interaction of 45-mer targets with the same mixture. A 7-base-long dangling end sequence with perfect homology to another single-stranded background DNA sequence was sufficient to produce a cross-hybridization signal that was as strong as the signal obtained by the probe-target duplex itself. Gibbs free energy between the target and a well-defined background was found to be a better indicator of hybridization signal intensity than the sequence or length of the dangling end alone. The dangling end (Gibbs free energy of -7.6 kcal/mol) was found to be significantly more prone to target-background interaction than the surface-proximal tail (Gibbs free energy of -64.5 kcal/mol). This study underlines the need for careful target preparation and evaluation of signal intensities for diagnostic arrays using 16S rRNA and other gene targets due to the potential for target interaction with a complex background.
Stedtfeld
Robert D
RD
Department of Civil and Environmental Engineering, Michigan State University, East Lansing, MI 48824, USA.
Wick
Lukas M
LM
Baushke
Samuel W
SW
Tourlousse
Dieter M
DM
Herzog
Amanda B
AB
Xia
Yongmei
Y
Rouillard
Jean Marie
JM
Klappenbach
Joel A
JA
Cole
James R
JR
Gulari
Erdogan
E
Tiedje
James M
JM
Hashsham
Syed A
SA
D001419:Bacteria / Q000737:chemistry / Q000235:genetics*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics; D001483:Base Sequence; D004269:DNA, Bacterial / Q000737:chemistry*; D020459:Genes, rRNA; D006801:Humans; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D015345:Oligonucleotide Probes / Q000737:chemistry; D016133:Polymerase Chain Reaction; D012336:RNA, Ribosomal, 16S / Q000235:genetics*; D013816:Thermodynamics
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D015345:Oligonucleotide Probes; D012336:RNA, Ribosomal, 16S