Enhanced nucleic acid capture and flow cytometry detection with peptide nucleic acid probes and tunable-surface microparticles. 2003

Darrell P Chandler, and Ann E Jarrell
Biochip Technology Center, Argonne National Laboratory, 9700 South Cass Ave., IL 60439, USA. dchandler@anl.gov

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.

UI MeSH Term Description Entries
D008832 Microchemistry The development and use of techniques and equipment to study or perform chemical reactions, with small quantities of materials, frequently less than a milligram or a milliliter.
D009693 Nucleic Acid Hybridization Widely used technique which exploits the ability of complementary sequences in single-stranded DNAs or RNAs to pair with each other to form a double helix. Hybridization can take place between two complimentary DNA sequences, between a single-stranded DNA and a complementary RNA, or between two RNA sequences. The technique is used to detect and isolate specific sequences, measure homology, or define other characteristics of one or both strands. (Kendrew, Encyclopedia of Molecular Biology, 1994, p503) Genomic Hybridization,Acid Hybridization, Nucleic,Acid Hybridizations, Nucleic,Genomic Hybridizations,Hybridization, Genomic,Hybridization, Nucleic Acid,Hybridizations, Genomic,Hybridizations, Nucleic Acid,Nucleic Acid Hybridizations
D009696 Nucleic Acids High molecular weight polymers containing a mixture of purine and pyrimidine nucleotides chained together by ribose or deoxyribose linkages. Nucleic Acid,Acid, Nucleic,Acids, Nucleic
D004269 DNA, Bacterial Deoxyribonucleic acid that makes up the genetic material of bacteria. Bacterial DNA
D005434 Flow Cytometry Technique using an instrument system for making, processing, and displaying one or more measurements on individual cells obtained from a cell suspension. Cells are usually stained with one or more fluorescent dyes specific to cell components of interest, e.g., DNA, and fluorescence of each cell is measured as it rapidly transverses the excitation beam (laser or mercury arc lamp). Fluorescence provides a quantitative measure of various biochemical and biophysical properties of the cell, as well as a basis for cell sorting. Other measurable optical parameters include light absorption and light scattering, the latter being applicable to the measurement of cell size, shape, density, granularity, and stain uptake. Cytofluorometry, Flow,Cytometry, Flow,Flow Microfluorimetry,Fluorescence-Activated Cell Sorting,Microfluorometry, Flow,Cell Sorting, Fluorescence-Activated,Cell Sortings, Fluorescence-Activated,Cytofluorometries, Flow,Cytometries, Flow,Flow Cytofluorometries,Flow Cytofluorometry,Flow Cytometries,Flow Microfluorometries,Flow Microfluorometry,Fluorescence Activated Cell Sorting,Fluorescence-Activated Cell Sortings,Microfluorimetry, Flow,Microfluorometries, Flow,Sorting, Fluorescence-Activated Cell,Sortings, Fluorescence-Activated Cell
D001407 Bacillus A genus of BACILLACEAE that are spore-forming, rod-shaped cells. Most species are saprophytic soil forms with only a few species being pathogenic. Bacillus bacterium
D012329 RNA, Bacterial Ribonucleic acid in bacteria having regulatory and catalytic roles as well as involvement in protein synthesis. Bacterial RNA
D012336 RNA, Ribosomal, 16S Constituent of 30S subunit prokaryotic ribosomes containing 1600 nucleotides and 21 proteins. 16S rRNA is involved in initiation of polypeptide synthesis. 16S Ribosomal RNA,16S rRNA,RNA, 16S Ribosomal,Ribosomal RNA, 16S,rRNA, 16S
D015341 Nucleic Acid Probes Nucleic acid which complements a specific mRNA or DNA molecule, or fragment thereof; used for hybridization studies in order to identify microorganisms and for genetic studies. Acid Probes, Nucleic,Probes, Nucleic Acid
D020135 Peptide Nucleic Acids DNA analogs containing neutral amide backbone linkages composed of aminoethyl glycine units instead of the usual phosphodiester linkage of deoxyribose groups. Peptide nucleic acids have high biological stability and higher affinity for complementary DNA or RNA sequences than analogous DNA oligomers. Peptide Nucleic Acid,Acid, Peptide Nucleic,Acids, Peptide Nucleic,Nucleic Acid, Peptide,Nucleic Acids, Peptide

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