Abstract:

Objective To reduce sampling frequency and shorten deafness gene detection period in detecting deafness gene by time-of-flight mass spectrometry with discarded dry blood spot generated from newborn screening. Methods The DNA extracting method of boiling discarded dry blood spot with alkaline water was optimized by adjusting the pH value and the dosage of alkaline water. Results When extracting DNA from discarded dry blood spot by alkaline water with the pH values of 9,10,11 and 12,all the 20 deafness gene were accurately detected,and the concentration of DNA was positively correlated with the pH value of alkaline water（r=0.906,P=0.094）,however,the DNA extracted from alkaline water with pH12 was difficult to be detected,as the peak value got sharply decreased. When extracting DNA from discarded dry blood spot by alkaline water with the pH value of 11 and the volumes of 60,70,80 and 90 μL,the concentration of DNA was negatively correlated with the volume of alkaline water（r=-0.988,P=0.012）,and the genetic locus of 547G>A in one samples extracted from 90 μL alkaline water with the pH value of 11 was failed to be detected,while the concentration of DNA extracted from discarded dry blood spot with 80 μL alkaline water with the pH value of 11 was close to those extracted from fresh dry blood spot（t=0.035,P=0.974）,and all the 20 genetic locuses related to deafness were accurately detected as well. Conclusions Good detection results could be obtained when extracting DNA from discarded dry blood spot through 80 μL alkaline water with the pH value of 11,and this study provides a reference on the construction of automatic detection platform for both newborn screening of genetic metabolic disease and deafness gene detection.

Key words: Dry blood spot, Newborn screening for genetic and metabolic diseases, Deafness, Extraction