Single-nucleotide polymorphism and mutation identification by the nanogen microelectronic chip technology.
The present chapter describes a microarray technology developed by Nanogen Inc., for the identification of DNA variations based on the use of microelectronics. The NMW 1000 NanoChip Molecular Biology Workstation allows the active deposition and concentration of charged biotinylated molecules on designated test sites. The DNA at each pad is then hybridized with specific oligonucleotide probes, complementary to normal or mutant sequences, that labeled with Cy3 or Cy5 dyes, respectively. The array is imaged, and fluorescence signals are scanned, monitored, and quantified by highly developed, digital image-processing procedures. The experimental steps to be performed for the development and execution of a microchip assay are described. Attention is focused on the fundamental aspects of probe design, and guidelines and useful suggestions are given. Protocols for sample preparation, addressing, reporting, and data analysis are also detailed.
Ferrari
Maurizio
M
Unit of Genomics for Diagnosis of Human Pathologies, Istituto di Ricovero e Cura a Carattere Scientifico Osedale San Raffaele, Milan, Italy.
Cremonesi
Laura
L
Bonini
Pierangelo
P
Foglieni
Barbara
B
Stenirri
Stefania
S
D001483:Base Sequence; D004252:DNA Mutational Analysis / Q000295:instrumentation / Q000379:methods; D006801:Humans; D025202:Molecular Diagnostic Techniques; D008969:Molecular Sequence Data; D009154:Mutation; D036103:Nanotechnology; D009690:Nucleic Acid Conformation; D020411:Oligonucleotide Array Sequence Analysis / Q000295:instrumentation / Q000379:methods; D020641:Polymorphism, Single Nucleotide