Genomic DNA was amplified about 5 billion-fold from single, flow-sorted bacterial cells by the multiple displacement amplification (MDA) reaction, using 29 DNA polymerase. A 662-bp segment of the 16S rRNA gene could be accurately sequenced from the amplified DNA. MDA methods enable new strategies for studying nonculturable microorganisms.The multiple displacement amplification (MDA) reaction uses the 29 DNA polymerase and random primers to amplify DNA templates (1-3, 5-6). Amplification from small specimens has enabled novel research approaches (reviewed in reference 7), including genetic analysis of single blastomeres for use in preimplantation diagnosis of embryos (4). A method to amplify genomic DNA from nonculturable bacteria would allow direct analysis of virtually any microbe. We demonstrate here the use of MDA to achieve several-billion-fold amplification of genomic DNA from a single bacterium. MDA could be used for a wide range of approaches for discovery of new species, population and polymorphism analysis, diagnostics, and rapid detection of pathogens.As a test case, E. coli cells (ATCC 10798; K-12 strain) were isolated (fluorescence-activated cell sorter Vantage flow cytometer [Becton Dickinson] using CellQuest and CytoCount softwares). To demonstrate proficiency in flow sorting, 180 putative cells were collected and vigorously vortexed in 10 l phosphate-buffered saline to separate cells in the event that more than one cell was obtained, and the number of CFU was determined (Fig.

Regional variations in the expression of a striatal enriched protein tyrosine phosphatase called STEP were studied in the adult rat brain by a combination of immunocytochemistry, lesion studies, Western blotting, and in situ hybridization. Monoclonal antibodies generated against STEP identified multiple polypeptides of M(r) 46, 37, 33 and a doublet of M(r) 64–66 kDa on Western blots. Although the three STEP immunoreactive bands with lower molecular weights were enriched in cytosolic fractions, the 64–66 kDa doublet was enriched in membrane fractions. All of the immunoreactive forms were abundant in the caudate-putamen and were present in lower amounts or were undetectable in other brain regions. In substantia nigra, the M(r) 64–66 kDa doublet was not detected but bands with M(r) 46, 37, and 33 kDa were present. Immunocytochemical and lesion experiments demonstrated that the cytosolic STEP isoforms present in the substantia nigra are in presynaptic axons originating from the projection neurons of the caudate putamen, which innervate this structure. Additional in situ hybridization studies showed that STEP mRNA expression patterns correlate with the patterns of immunocytochemical staining. These findings indicate that there are multiple polypeptide isoforms of STEP enriched in the basal ganglia and related structures which differ in terms of their intracellular locations and functional roles.

Immunohistochemistry is a method that can provide complementary diagnostic and prognostic information to morphological observations and soluble assays. Sensitivity, specificity, or requirements for arduous sample preparation or signal amplification procedures often limit the application of this approach to routine clinical specimens. Rolling circle amplification (RCA) generates a localized signal via an isothermal amplification of an oligonucleotide circle. The application of this approach to immunohistochemistry could extend the utility of these methods to include a more complete set of immunological and molecular probes. RCA-mediated signal amplification was successfully applied to the sensitive and specific detection of a variety of cell surface antigens (CD3, CD20, and epithelial membrane antigen) and intracellular molecules (vimentin and prostate-specific antigen) within a variety of routinely fixed specimens, as well as samples prepared for flow cytometry. RCA technology, which has an intrinsically wide dynamic range, is a robust and simple procedure that can provide a universal platform for the localization of a wide variety of molecules as a function of either antigenicity or nucleic acid sequence. The use of RCA in this way could enhance the use of markers of current interest as well as permit the integration of emerging information from genomics and proteomics into cell- and tissue-based analyses.

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