Genotyping array protocols

eagle-i ID

Resource Type

Properties

1. Resource Description

   "Protocols vary depending upon the array being run, so it is wise to check with the IMC to see how the following applies to a specific product. DNA is prepared by standard high quality protocols. 200 to 750 ng is needed for each sample, depending upon the size of the array (1). The DNA is denatured and then neutralized for amplification. The denatured DNA is isothermally amplified several thousandfold using a proprietary Illumina protocol. The amplified product is fragmented by a controlled enzymatic process that does not require gel electrophoresis. Overfragmentation is avoided by using end-point fragmentation. Fragmented DNA is recoved by isopropanol precipitation. The fragmented DNA is then hybridized to the 50-mer probes of the BeadChip. Unhybridized and non-specific DNA is washed from the array. Single base extension of the oligos on the BeadChip incorporates modified nucleotides at the site of the single nucleotide polymorphism. The labeled nucleotides include biotin-labeled ddCTP and ddGTP and 2,4-dinitrophenol (DNP) -labeled ddATP and ddUTP. Following additional washing to remove unincorporated nucleotide, the arrays are stained with Alexa555 and Alexa 647 using a dual color, orthogonal, multi-layer immunohistochemical sandwich assay (2). Stained arrays are scanned using the Illumina BeadReader microarray scanner. The data is consolidated using Illumina's GenomeStudio software."

2. Uses

   GenomeStudio

3. Uses

   Illumina BeadReader scanner

4. Used by

   Gene Profiling Shared Resource Core Laboratory