Enhanced nucleic acid capture and flow cytometry detection with peptide nucleic acid probes and tunable-surface microparticles.
New methods for automated, direct nucleic acid purification and detection are required for the next generation of unattended environmental monitoring devices. In this study we investigated whether tunable-surface bead chemistry and peptide nucleic acids (PNA) could enhance the recovery and detection of intact rRNA in both test tube and automated suspension array hybridization formats. Intact rRNA was easily captured and detected on PNA-coated Lumavidin beads from 0.1 ng total RNA with a 15-min hybridization in pH 7 buffer, representing 1.7 x 10(3) cell equivalents of total RNA. DNA-conjugated beads in pH 5 hybridization buffer required an overnight hybridization to achieve a detectable signal at 0.1 ng target RNA. Standard DNA hybridization conditions (pH 7) were one order of magnitude less sensitive than the tunable-surface (pH 5) condition. The PNA-conjugated particles were 100x more sensitive than the tunable-surface DNA particles in the automated format, with a detection limit of 0.1 ng total RNA. The detection limits for total RNA on PNA-conjugated microparticles is immediately conducive to the detection and characterization of microorganisms in low-biomass environments or to the identification of rare sequences in a complex sample mixture, without using PCR.
Chandler
Darrell P
DP
Biochip Technology Center, Argonne National Laboratory, 9700 South Cass Ave., IL 60439, USA. dchandler@anl.gov
Jarrell
Ann E
AE
D001407:Bacillus / Q000235:genetics; D004269:DNA, Bacterial / Q000032:analysis; D005434:Flow Cytometry / Q000379:methods*; D008832:Microchemistry / Q000379:methods; D009693:Nucleic Acid Hybridization; D015341:Nucleic Acid Probes / Q000032:analysis*; D009696:Nucleic Acids / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D020135:Peptide Nucleic Acids / Q000032:analysis*; D012329:RNA, Bacterial / Q000032:analysis; D012336:RNA, Ribosomal, 16S / Q000032:analysis
D004269:DNA, Bacterial; D015341:Nucleic Acid Probes; D009696:Nucleic Acids; D020135:Peptide Nucleic Acids; D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S