Abstract:

OBJECTIVE: Pyrosequencing was applied to study the effect of whole genome amplification (WGA) on the fidelity of DNA methylation. METHODS：EpiTect Whole Bisulfitome Kit was used to perform DNA amplification after whole genome bisulfite modification of L02，A549 and HCT116 cells. The DNA methylation of three specific genes (MGMT，GSTP1，P16) and LINE 1 sequence were analyzed by pyrosequencing. RESULTS：The DNA amplification obtained was 246 folds higher in DNA content and > 1 000 bp fragment length which met the basic requirements of DNA methylation detection. The DNA methylation level of LINE1 did not change after WGA，indicating that WGA had no effect on the whole genome methylation. However，the change of specific DNA methylation after WGA correlated with their original methylation level. When the original methylation rate was higher than 60%，the methylation condition of three genes remained stable after WGA. There was significant change with irregular trend of DNA methylation level after WGA when the initial DNA methylation ranged between ≥20%-60%. DNA methylation rate of three genes declined significantly in all cells when the initial DNA methylation level was lower than 20%. In addition，pyrosequencing result showed that the effect of WGA on genes CpG sites methylation level was similar to that on its mean methylation rate. CONCLUSION：WGA technology was suitable for the study of whole genome methylation and specific gene with higher methylation rate (≥60%). However，we must take caution for using the WGA technology when the rate of DNA methylation was relative low (< 60%).

Key words: DNA methylation, whole genome amplification, pyrosequencing, fidelity