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.

Rad51 and Rad54 proteins are important for the repair of double-stranded DNA (dsDNA) breaks by homologous recombination in eukaryotes. Rad51 assembles on single-stranded DNA (ssDNA) to form a helical nucleoprotein filament that performs homologous pairing with dsDNA; Rad54 stimulates this pairing substantially. Here, we demonstrate that Rad54 acts in concert with the mature Rad51-ssDNA filament. Enhancement of DNA pairing by Rad54 is greatest at an equimolar ratio relative to Rad51 within the filament. Reciprocally, the Rad51-ssDNA filament enhances both the dsDNA-dependent ATPase and the dsDNA unwinding activities of Rad54. We conclude that Rad54 participates in the DNA homology search as a component of the Rad51-nucleoprotein filament and that the filament delivers Rad54 to the dsDNA pairing locus, thereby linking the unwinding of potential target DNA with the homology search process.

The flap endonuclease, FEN1, plays a critical role in DNA replication and repair. Human FEN1 exhibits both a 5' to 3' exonucleolytic and a structure-specific endonucleolytic activity. On primer-template substrates containing an unannealed 5'-tail, or flap structure, FEN1 employs a unique mechanism to cleave at the point of annealing, releasing the 5'-tail intact. FEN1 appears to track along the full length of the flap from the 5'-end to the point of cleavage. Substrates containing structural modifications to the flap have been used to explore the mechanism of tracking. To determine whether the nuclease must recognize a succession of nucleotides on the flap, chemical linkers were used to replace an interior nucleotide. The nuclease could readily traverse this site. The footprint of the nuclease at the time of cleavage does not extend beyond 25 nucleotides on the flap. Eleven-nucleotide branches attached to the flap beyond the footprinted region do not prevent cleavage. Single- or double-thymine dimers also allow cleavage. cis-Platinum adducts outside the protected region are moderately inhibitory. Platinum-modified branch structures are completely inert to cleavage. These results show that some flap modifications can prevent or inhibit tracking, but the tracking mechanism tolerates a variety of flap modifications. FEN1 has a flexible loop structure through which the flap has been proposed to thread. However, efficient cleavage of branched structures is inconsistent with threading the flap through a hole in the protein.

Background: Rolling circle amplification of ligated probes is a simple and sensitive means for genotyping directly from genomic DNA. SNPs and mutations are interrogated with open circle probes (OCP) that can be circularized by DNA ligase when the probe matches the genotype. An amplified detection signal is generated by exponential rolling circle amplification (ERCA) of the circularized probe. The low cost and scalability of ligation/ERCA genotyping makes it ideally suited for automated, high throughput methods.