Preparation of genomic DNA and direct sequencing
Genomic DNA was prepared from peripheral blood samples using a Puregene blood DNA kit (Gentra, Minneapolis, MN, USA), following the manufacturer's protocol.
To identify polymorphisms in the promoter of the AKT2 and AKT3 genes, human genomic DNA was isolated from the whole blood of 24 lung cancer patients for direct sequencing, and the promoter region of the AKT2 and AKT3 genes was amplified. Polymerase chain reaction (PCR) amplifications were performed on a PTC-225 Peltier Thermal cycler (MJ Research Inc., Waltham, MA, USA) using Ampli-TagGold (Roche, Branchburg, NJ, USA). All amplifications were performed using 35 cycles of 30 s at 95℃, 1 min at 64℃, and 1 min at 72℃, followed by a single 10-min extension at 72℃. PCR products were purified using the Montage PCR96 Cleanup kit (Millipore, Bedford, MA, USA) and eluted in 20 µL of nuclease-free H2O. DNA cycle sequencing was carried out using the BigDye Terminator V 3.1 Cycle Sequencing kit (Perkin Elmer, Foster City, CA, USA). Multiscreen SEQ 384-well filter plates were used for dye removal, and sequences were then analyzed on an Applied Biosystems 3700 (ABI, Foster City, CA, USA). All polymorphisms and sequence alignments were analyzed using Polyphred.