Single Molecule Biology

Single Molecule Biology

2009, Pages 289-316
Single Molecule Biology

Chapter 10 - Single Molecule Microarray Analysis

https://doi.org/10.1016/B978-0-12-374227-8.00010-9Get rights and content

Publisher Summary

In recent years, microarrays have become a standard tool not only for expression analysis in molecular biology and genomic research but also for pharmacogenomics and infectious and genetic disease and cancer diagnostics. This chapter describes the basics of microarray technology and microarrays, including DNA microarrays and protein microarrays and highlights successful applications of single molecule detection on microarrays to pursue mRNA expression profiling, DNA methylation analysis, DNA fragment sizing, sequencing of individual DNA molecules, and physical mapping of DNA molecules using the molecular combing technique. The linear dependence of the fluorescence brightness obtained from a single biomolecule with the fragment size has been used for determination of the DNA fragment sizes present in the sample. Rapid data analysis, in combination with microfluidic devices, enabled the construction of a single molecule DNA sorting device. Detailed sequence information directly on individual high-molecular-weight DNA molecules can be obtained by physical mapping approaches using the “molecular combing” technique, which involves covalent binding of individual DNA molecules to a solid substrate and elongating them by a receding meniscus of solute. Several approaches have been developed for single molecule sequencing. Quantification of methylation of genomic DNA hybridized to an oligonucleotide microarray has been performed by specifically labeling 5'-methyl-cytosine (5'mC) using monoclonal anti-5'mC antibody and a secondary Cy3-conjugated antibody. Furthermore, the chapter explores the limits of sensitivity reachable by microarray technology, discussing in particular the applications based on the detection of single nucleic acid molecules on a microarray format, which offer additional access to new types of information not obtainable from ensemble-based studies.

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