Isolation of human genomic DNA from archival dried blood spots for neonatal disease screening and its application to methylation detection
10.3760/cma.j.issn.2095-428X.2015.08.014
- VernacularTitle:新生儿疾病筛查血片微量DNA提取方法的建立及其在甲基化检测中的应用
- Author:
Qiuping CHEN
;
Xianwei CUI
;
Lianghui YOU
;
Chenbo JI
;
Xirong GUO
- Publication Type:Journal Article
- Keywords:
Archival dried blood spot;
Isolation of human genomic DNA;
Methylation;
Methylation-specific polymerase chain reaction
- From:
Chinese Journal of Applied Clinical Pediatrics
2015;30(8):615-618
- CountryChina
- Language:Chinese
-
Abstract:
Objective To establish an effective DNA isolation method for neonatal disease screening,so as to explore its application to the methylation detection.Methods The 20 dried blood spots samples were randomly divided into 2 groups according to the gender:the traditional method group (n =10) and the improved kit method group(n =10).The DNA quality was evaluated based on its concentration,integrity and whether it could be used in polymerase chain reaction (PCR).These DNA samples with or without bisulfite treatment were used as template in the methylation-specific polymerase chain reaction (MSP).The methylation levels of Leptin and tumor necrosis factor-α (TNF-α) gene promoter region were detected.Results DNA concentration of the improved kit method [(5.70 ± 0.81) mg/L] was significantly higher than that of the traditional method [(3.50 ± 0.45) mg/L] (t =2.79,P ＜ 0.05),and biochemical analyzer analysis showed a better DNA integrity.Agarose gel electrophoresis revealed that 18S gene fragment could be successfully amplified by PCR method,suggesting its potential application to PCR study.MSP results showed different DNA methylation levels of Leptin and TNF-α genes promoter regions from various samples.Conclusions The improved kit method can effectively extract DNA from dried blood spots samples,and these DNA can be used in methylation research.The study can provide a new research direction and technical method to reveal the pathogenesis of disease from the perspective of DNA methylation.
