Bisulfite-sequencing PCR (BSP)
The DNA from the MSP results was subjected to sodium bisulfite treatment
via a DNA bisulfite kit (Qiagen, Beijing, China) according to the
manufacturer’s instructions. After bisulfite conversion, the DNA was
purified and recovered via the SanPrep Column DNA Gel Extraction Kit
(Sangon, Shanghai, China). Primers for bisulfite pyrosequencing were
designed to target the specific region of interest and were utilized
with 3730 sequencing analyzers. The primers for the TP53BP2 methylation
reaction were as follows: TP53BP2 forward: 5′-AACTTCACGGTGGGTTTCAAGC-3′;
TP53BP2 reverse: 5′-GTGCAGGCCTGAGCCTTCT GGC-3′.