500k Genotyping Arrays

  • The Affymetrix GeneChip® 500K SNP oligonucleotide microarray chip pair, which is comprised of a 250K Sty I array and a 250K Nsp I array, is designed to discriminate alleles at approximately 500,000 dimorphic positions in the human genome. These microarrays have been particularly useful in cancer research for carrying out genome-wide analysis of allelic changes (including deletions, amplifications, and gene conversions) in tumors. They are also useful in linkage analysis. The DNA from a fresh or frozen source can be analyzed on the SNP microarrays. The DNA must be double-stranded, from blood or cell lines, and free of PCR inhibitors. In addition, the DNA must not be degraded or contaminated with other genomic DNA sources. Experimental turn-around times are usually four business days.

    Isolation
    Our analysis begins with genomic DNA that is provided by an investigator. The 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. Using the SDS/ProK digestion, phenol-chloroform extraction, Microcon® or Centricon® (Millipore) ultrapurification and concentration has been tested at Affymetrix and produces successful isolation for genomic DNA from cell lines. The QIAmp® Blood Maxi kit from QIAGEN has also been tested at Affymetrix and successfully isolates genomic DNA. Please contact our core facility if you are uncertain as to whether a DNA extraction method that you are considering is suitable.

    Quantity
    Investigators are asked to provide 500ng of genomic DNA (or 250ng if a sample will be hybridized to only one of the 250K arrays) at a concentration of 50ng/µL, from each specimen. The SNP protocol requires 5µL of DNA concentrated at 50ng/µL for a total of 250ng per array. Documentation of sample quantities needs to be provided before experiments begin. Depending on the number of samples submitted, all sample quantities are rechecked on the NanoDrop 1000 Spectrophotometer or a plate reader. Once the quantity has been verified for each sample, your experiment will begin. If quantity is an issue for any sample, the investigator will be notified immediately to discuss the next course of action. Please provide an extra 200ng of genomic DNA in addition to the experimental quantity in order to account for the protocol checkpoints and any concentration variations, should your sample concentrations be off. If you are having trouble obtaining this amount, you may wish to try a procedure known as "whole genome amplification,” that has been shown to reliably amplify DNA by several orders of magnitude. This amplification does not alter the SNP determinations. For example, the Repli-G® kit from QIAGEN can be used to amplify as little as 10ng of starting material of genomic DNA.