We discuss a novel atomic force microscope-based method for identifying individual short DNA molecules ( 5000 bp) within a complex mixture by measuring the intra-molecular spacing of a few sequence-specific topographical labels in each molecule. microarrays and next-generation sequencing, but not without certain shortfalls and shortcomings . PCR and probe hybridization techniques, which rely on the assembly of highly specific molecular complexes, satisfy the high gain requirements, but suffer serious problems when used with high-complexity mixtures. In situations with many different targets present at low abundance, the kinetics of molecular complex formation is PF-562271 novel inhibtior usually unfavourable and many probe species are required. Typically, this diversity PF-562271 novel inhibtior leads to unacceptable cross-talk between probes or requires the use of secondary sorting methods to reduce the complexity of the sample. Microarrays and nextgen-sequencing technologies are relatively insensitive and require enzymatic amplification of low-abundance samples. The amplification process is slow, technically complex and distorts the relative abundance of species, particularly those with high sequence similarity (i.e. transcript variants, gene family members, and so on) . Nanotechnology-based single molecule approaches provide a competing approach to such applications requiring molecular recognition, thus opening new avenues to medical diagnostics, genetic assessments and pathogen detection. In this paper, we explore a novel, alternative method for identifying individual DNA molecules within a complex mixture, whereby the becomes the identifying probe, thereby avoiding many of the problems inherent in the established methods discussed above. In our approach, the backbone of each DNA molecule is usually decorated with a few topographical labels, introduced at nicking endonuclease recognition sites which are Rabbit Polyclonal to ACTL6A measured very precisely with atomic force microscopy (AFM), to form a pattern unique to that species. A key advantage of this approach is that the labelling chemistry is simple, highly parallel (a single label used for all molecules) and no amplification is required. We illustrate this method based on a self-labelling approach in the context of potential application to an important problem in molecular biology: identifying individual cDNA molecules in a low-abundance sample (e.g. single cell) for the purpose of gene-expression profiling. 2.?Material and methods 2.1. DNA labelling protocol DNA samples are PF-562271 novel inhibtior diluted in 1 NEBuffer 4 (50 mM potassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate, 1 mM dithriothreitol, pH 7.9; New England Biolabs) enzymatically tagged with 1 U nicking enzyme nt.BsmAI (New England Biolabs) for 1 h at 37C. Linearized and nicked DNA is usually spin-purified and eluted with purified water, pH 8.3, or 10 mM TrisCCl, pH 8.5 (Qiagen QIAquick Gel Extraction Kit). Sample concentration is determined by fluorometric quantitation (Qubit Fluorometer) before biotin incorporation. Biotin dUTP labelling at 3 ends is usually incorporated at nick sites through a terminal transferase reaction in 1 terminal transferase buffer (Roche), 5 mM CoCl2 (Roche), 0.05 mM Biotin-16-dUTP (Roche) and 20 U terminal transferase enzyme (New England Biolabs) for 1 h at 37C. The biotinylated DNAs are spin-purified and eluted with 10 mM TrisCCl, pH 8.5 (Qiagen QIAquick Gel Extraction Kit). For AFM visualization of tagged nick sites, approximately 1 g Streptavidin (New England Biolabs) is added to the biotinylated sample and incubated at room temperature for more than 2 h or overnight at 4C. For the experiments with lambda phage DNA, samples were prepared from N6-methyladenine-free lambda DNA (New England Biolabs), cut into 15 fragments in 1NEBuffer 4 and 5 U = approximately 3 N m?1 silicon probes (Nanosensors). Image resolution was 2 nm pixel?1. DNA contour lengths and streptavidin label locations were measured manually with NIH ImageJ. Classification of individual molecule as belonging to a particular species is accomplished using the same alignment algorithm as in the simulation (see 2.4.2), with the following exceptions: the universe of available hypotheses was limited to the 15 fragments known to be present in the mixture; to be conservative, the allowed label alignment precision and overall length measurement precision were taken as 4.