2.2. SNP selection, DNA extraction, target DNA amplified and genotyping

Three SNPs in CDH1 were selected in this research based on the following standard: prior evidence of association with CL/P and minor allele frequency of the SNP of at least 20% in the Chinese population in accordance with the HapMap data.

DNA was extracted from peripheral blood using an AxyPrep-96 DNA Isolation kit (Axygen Scientific, Santa Clara, CA) following the manufacturer's protocol for genetic analysis. The DNA pellet was dissolved in Tris-EDTA buffer, and its purity and concentration were identified by the spectrophotometric measurement of the absorbance at 260 and 280 nm.

Through multiplex polymerase chain reactions (PCRs), the target DNAs were amplified. PCRs were performed in 15-μL solutions containing 1 μL DNA, 1.5 μL of 10× buffer, 1.5 μL MgCl₂, 0.3 μL dNTP, 0.3 μL of Taq polymerase (Fermentas, Canada), and 0.15 μL of each primer. PCR conditions were as follows: an initial 94°C for 3 minutes, then 35 cycles of 94°C for 15 seconds, 55°C for 15 seconds, 72°C for 30 seconds, and a final at 72°C for 3 minutes.

Genotyping of the 3 SNPs was performed using the Snapshot mini-sequencing technique. Three microliter of the PCR product was purified with 0.8 μL of FastAP (Fermentas, Canada) and 0.2 μL of ExoI (Fermentas, Canada), and incubated at 37°C for 15 minutes in turn at 80°C for 15 minutes. The extension reactions were conducted in a total volume of 6 μL containing 1 μL of Snapshot Mix (Applied Biosystems), 2 μL of purified PCR product, and 0.2 μL of each extension primers. The cycling conditions were 96°C for 1 minute, then 30 cycles of 96°C for 10 seconds, 52°C for 5 seconds, and 60°C for 30 seconds. One microliter of the extension products were mixed with 9 μL of HIDI (Applied Biosystems), and then, the compound was denatured at 95°C for 3 minutes. Then, the 3 SNPs were analyzed using an ABI PRISM 3730 DNA Sequencer (Applied Biosystems). For the sake of quality control, reactions were conducted repeatedly in 10% of the samples at random for each SNP, and the results were the same.