Genotyping using Affymetrix arrays

Sample DNAs should not be highly degraded nor contain PCR inhibitors, such as high concentrations of heme or chelating agents. For each individual assayed, 250 ng of genomic DNA are digested separately with 10 U of XbaI or HindIII (New England BioLabs) in volumes of 20 µL for 2 hours at 37 °C. Following heat inactivation at 70 °C for 20 minutes, 0.25 µM of XbaI adaptor (5’-ATT ATG AGC ACG ACA GAC GCC TGA TCT-3’ and 5’phosphate ?CTA GAG ATC AGG CGT CTG TCG TGC TCA TAA-3’)(Affymetrix), or HindIII adaptor (5’-ATT ATG AGC ACG ACA GAC GCC TGA TCT-3’ and 5’phosphate ?AGC TAG ATC AGG CGT CTG TCG TGC TCA TAA-3’) (Affymetrix) are ligated to the digested DNAs with T4 DNA Ligase (New England BioLabs) in 25µL for 2 hours at 16°C. The ligations are stopped by heating to 70 °C for 20 minutes, and then diluted 4- fold with water. For each ligation reaction, two to three PCRs are run in order to generate > 40 µg of PCR products. Each PCR contains 10 µL of the diluted ligation reactions (25 ng of starting DNA) in 100 µL volumes containing 1.0µM of primer (5’-ATT ATG AGC ACG ACA GAC GCC TGA TCT-3’), 0.30 mM dNTPs, 1.0 mM MgSO4, 5 U Platinum® Pfx Polymerase (Invitrogen), PCR Enhancer (Invitrogen) and Pfx Amplification Buffer (Invitrogen). 30 cycles of PCRs are run with the following cycling program: 94 °C denaturation for 15 seconds, 60 °C annealing for 30 seconds, and 68 °C extension for 60 seconds. As a check, 3 µL of PCR products are visualized on 2% TBE agarose gels to confirm the size range of amplicons. The PCR products are purified over MinElute 96 UF PCR Purification plates (Qiagen), and recovered in 40 µL of EB buffer (Qiagen). PCR yields are measured by absorbance readings at 260 nm, and adjusted to a concentration of 40 µg per 45 µl. To allow efficient hybridization to 25-mer oligonucleotide probes, the PCR products are fragmented to < 100 bp with DNAse I. 0.20 U of DNAse I (Affymetrix) is added to 40 ug of purified PCR amplicons in a 55 µL volume containing Fragmentation Buffer (Affymetrix) for 35 minutes at 37 °C, foll owed by heat inactivation at 95 °C for 15 minutes. Fragmentation products are visualized on 4% TBE agarose gels. The 3’ ends of the fragmented amplicons are biotinlyated by adding 214 µM of a proprietary DNA labeling reagent (Affymetrix) using Terminal Deoxynucleotidyl Transferase (Affymetrix) in 70 µL volumes for 2 hours at 37 °C, followed by heat inactivation at 95 °C for 15 minutes.

Allele Specific Hybridization to Oligonucleotide Arrays

The fragmented and biotinylated PCR amplicons are combined with 11.5 µg/mL human Cot-1 (Invitrogen) and 115 µg/mL herring sperm (Promega) DNAs. The DNAs are added to a hybridization solution containing 2.69 M tetramethylamonium chloride (TMACl), 5.77 mM EDTA, 56 mM MES, 5 % DMSO, 2.5 X Denhardt’s solution, and 0.0115% Tween-20 in a final volume of 260 µL. The hybridization solution was heated to 95 °C for 10 minutes then placed on ice. After warming to 48 °C for 2 minutes, 200 µL of the hybridization solution is injected into cartridges housing the oligonucleotide arrays (Affymetrix GeneChip® 100K Mapping Set: 50K Array Xba 240 and 50K Array Hind 240). Hybridizations are carried out at 48 °C for 16 to 18 hours in a rotisserie rotating at 60 rpm. Following the overnight hybridization, the arrays are washed with 6XSSPE and 0.01% Tween-20 at 25 °C, then more stringently washed with 0.6X SSPE and 0.01% Tween-20 at 45 °C. Hybridization signals are generated in a three step signal amplification process: 10µg/mL streptavidin R-phycoerythrin (SAPE) conjugate (Molecular Probes) is added to the biotinylated targets hybridized to the oligonucleotide probes, and washed with 6X SSPE and 0.01% Tween-20 at 25 °C; followed by the addition of 5µg/mL biotinylated goat anti-streptavidin (Vector) to increase the effective number of biotin molecules on the target; and finally SAPE is added once again and washed extensively with 6X SSPE and 0.01% Tween-20 at 30 °C. The SAPE and antibody were added to arrays in 6X SSPE, 1X Denhardt’s solution and 0.01% Tween-20 at 25 °C for 10 minutes each. Following the final wash, the arrays are kept in Holding buffer (100mM MES, 1M [Na+], 0.01% Tween-20). The washing and staining procedures are run on Affymetrix fluidics stations. Arrays are scanned using GCS3000 scanners with AutoLoaders (Affymetrix). Scan images are processed to get hybridization signal intensity values using GCOS 2.0 software (Affymetrix). The DM genotype calling algorithm is implemented in GenoTyping Tools (GTT) (Affymetrix) and GDAS 3.0 (Affymetrix) analysis software.

(from Hajime Matsuzaki, Shoulian Dong, Halina Loi, Xiaojun Di, Guoying Liu, Earl Hubbell, Jane Law, Tam Berntsen, Monica Chadha, Henry Hui, Geoffrey Yang, Giulia C Kennedy, Teresa A Webster, Simon Cawley, P Sean Walsh, Keith W Jones, Stephen P A Fodor & Rui Mei. Genotyping over 100,000 SNPs on a pair of oligonucleotide arrays.

Nature Methods  1, 109 - 111 (2004) . PubMed ID: 15782172)