Avoidance of nonspecific hybridization by employing oligonucleotide micro-arrays generated from hydrolysis polymerase chain reaction probe sequences.
We have developed a low-density oligonucleotide-based micro-array where 5'-end-tethered capture probe sequences were derived from Primer Express software. The capture probes represent hydrolysis probe sequences devoid of any fluorochromes and were shown to retain hybridization binding specificity to their amplicons; hybridization specificity was retained independent to probe sequences. This procedure allowed the specificity of each capture probe to be verified using real-time polymerase chain reaction (PCR) in the presence of nucleic acid sequences typically expected to be present within a sample and therefore has reduced possibility of nonspecific hybridization when used in a micro-array format. We propose that specificity-validated probes are applied to form a micro-array for the purpose of general target screening, with incumbent multiparallelization and cost and time savings. However, if required, the subset of probe sequences of interest can be used for quantitative assessment in conventional real-time PCR.
Greiner
Oliver
O
Functional Genomics Unit, Division of Oncology, University Children's Hospital of Zurich, August Forel Strasse 1, Zurich, CH-8008, Switzerland.
Day
Philip J R
PJ
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D006868:Hydrolysis; D009693:Nucleic Acid Hybridization / Q000379:methods*; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods* / Q000592:standards; D015345:Oligonucleotide Probes / Q000138:chemical synthesis / Q000592:standards*; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D012984:Software; D013296:Streptococcus pneumoniae / Q000235:genetics; D013301:Streptolysins / Q000235:genetics
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D015345:Oligonucleotide Probes; D013301:Streptolysins; C032290:plY protein, Streptococcus pneumoniae