|
|  |
500K Genotyping (SNPs) 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.
|
|
contact us 
Print
This Page
