Genomics Studies - Biomedical Informatics in Translational Research - page 63

Figure 5.1 GeneChip mapping assay overview. (Courtesy of Affymetrix.)

program is run to amplify DNA fragments that are between 200 and 1,100 base
pairs (bp) long. This step reduces the DNA complexity and produces enough DNA
for hybridization. The PCR products are purified using the QIAquick PCR purifica-
tion kit (Qiagen), quantified, fragmented using DNase1, and then labeled with bio-
tin. The reduced size of DNA fragments ensures that hybridization to the 25mers
probes is achieved more efficiently.
Labeled DNA fragments are then injected into GeneChip arrays for overnight
hybridization and then washed and stained in an Affymetrix Fluidic station 450
using preinstalled Affymetrix protocols. The final step is to scan the arrays on an
Affymetrix Scanner 3000 with default settings. The scanned images are analyzed
using GeneChip Genotyping Analysis software (GTYPE), which generates
Microsoft Excel files that include the calls for each marker. The raw data file can be
imported into other software such as Merlin and Viria (Agilent) for further analysis

5.2.2.4 QA/SOP Issues
The most important factor that affects the quality of SNP results is the quality of the
sample DNA. Both sequencing and primer extension technologies have been
applied in research for a long time and are relatively mature and easy to accomplish.
Most primers designed for genotyping are well studied, and once the optimized con-
dition for these primers been achieved, the failure rate is relatively small. However,
running the Affymetrix GeneChip genotyping arrays requires precise and careful
handling.
Three quality control/quality assurance (QC/QA) steps are used to check the
quality of the reactions. The first step occurs after the PCR step, at which time an
aliquot is run on an agarose gel. The resulting lengths of the PCR products must be
in the range of 200 to 1,100 bp. The second QC/QA step occurs after PCR product
purification and elution when the product is quantified and normalized; this step is
critical for hybridization. Purified and diluted PCR products are quantified by mea-
suring the optical density (OD) at 260, 280, and 320 nm. A typical average sample
OD260 is 0.5 to 0.7. The OD260/OD280 ratio should be between 1.8 and 2. The
sample is discarded if this metric falls outside of this range. The OD320 measure-