DNA Isolation

The analysis in the Core begins with genomic DNA that is provided by an investigator. DNA extracted from either fresh or frozen tissue is suitable for analysis on the SNP arrays; at this point we are unaware of any particular DNA isolation protocol that produces DNA of a more suitable quality than another.

DNA Amount

Please provide 750 ng of genomic DNA at a concentration of 50 ng/µl.

Arrays

The SNP 6.0 array interrogates 900,600 dimorphic SNP loci using 1 probe sequence per allele with each probe repeated at 3-4 locations on the array.  Also present on the array are 946,000 probes that correspond to non-SNP (“non-polymorphic” probes) locations in the human genome.  202,000 of the “non-polymorphic” probes target 5,677 regions of documented germline polymorphic copy number variation; the remaining 744,000 “non-polymorphic” probes correspond to sites distributed across the human genome.

Laboratory Process

1. The SNP 6.0 analysis begins with 2 aliquots of 250 ng of DNA in each: one is cleaved with StyI; the other is cleaved with NspI.
2. Linkers are ligated to the restricton fragments using T4 DNA ligase. The linkers provide a primer site for the subsequent PCR reaction.
3. The fragments are amplified using Taq polymerase. The PCR products are pooled so that the NspI products are combined with the StyI products.  The samples are purified from the primers and free nucleotides by ultrafiltration. They are then quantified spectrophotometrically, and are also assayed using size fractionation on a microfluidics device to determine whether the size distribution of products is as expected.
4. The purified PCR products are fragmented by DNAse I in order to provide 3' hydroxyl groups for subsequent labeling, and to facilitate the subsequent hybridization step.
5. The fragmented PCR products are labeled with a single biotin at each free 3'OH using terminal deoxynucleotidyl transferase and a biotinylated dideoxy nucleoside triphosphate.
6. The biotinylated fragments are added to a hybridization solution containing a biotinylated control oligonucletide (for quality control), and hybridized to a microarray chip overnight at 50°C.
7. The chips are then transferred to a fluidics instrument that performs washes to remove DNA that has not hybridized to its complementary oligonucleotide probe. The bound DNA is then fluorescently labeled using phycoerythrin-conjugated streptavidin (SAPE) followed by biotinylated anti-streptavidin, followed by SAPE.
8. Each DNA bound at its complementary oligonucleotide is excited using a confocal laser scanner, and the positions and intensities of the fluorescent emissions are captured. These measures provide the basis of subsequent biostatistical analysis. The data is transferred to a database from which it can be downloaded via a password-protected URL.