Makowski G S, Davis E L, Nadeau F, Hopfer S M
Department of Laboratory Medicine, University of Connecticut School of Medicine, Farmington 06030, USA.
Ann Clin Lab Sci. 1998 Jul-Aug;28(4):254-9.
This study evaluated two deoxyribonucleic acid (DNA) extraction methods for Guthrie card bloodspots. Our water-based extraction technique was compared with a commercial kit (GFX extraction system). In contrast to our nondenaturing method, the GFX system utilizes chaotropes to extract DNA. Extracted DNA is subsequently purified by selective elution from a glass fiber matrix. To evaluate DNA extraction, polymerase chain reaction (PCR) for a 491 bp region encoding the cystic fibrosis delta F508 mutation was performed and PCR products electrophoresed on polyacrylamide gels and stained with ethidium bromide. Amplification of GFX-extracted DNA required low sample volume (1 to 5 microL) indicating the presence of residual PCR inhibitors. The GFX volumes (1/20 to 1/4 punch) were comparable to our standard conditions and represented 0.16-0.80 microL whole blood (20 to 40 ng DNA). The GFX-extracted DNA was found to be stable (6 mo, -15 degrees C). Performance time for the GFX method was less than our standard water-based extraction (about 30 min), but more costly. The GFX extraction kit was adaptable for limited sample (i.e., extraction of a 3 mm punch yields 20 amplifications). The GFX extraction kit provides a useful means to standardize Guthrie card DNA extraction.
本研究评估了用于干血滤纸片的两种脱氧核糖核酸（DNA）提取方法。我们的水基提取技术与一种商业试剂盒（GFX提取系统）进行了比较。与我们的非变性方法不同，GFX系统利用离液剂来提取DNA。随后，通过从玻璃纤维基质中选择性洗脱来纯化提取的DNA。为了评估DNA提取效果，针对编码囊性纤维化ΔF508突变的491 bp区域进行了聚合酶链反应（PCR），并将PCR产物在聚丙烯酰胺凝胶上进行电泳，用溴化乙锭染色。对GFX提取的DNA进行扩增所需的样本量较低（1至5微升），这表明存在残留的PCR抑制剂。GFX的用量（1/20至1/4打孔量）与我们的标准条件相当，相当于0.16 - 0.80微升全血（20至40纳克DNA）。发现GFX提取的DNA是稳定的（6个月，-15摄氏度）。GFX方法的操作时间比我们的标准水基提取方法短（约30分钟），但成本更高。GFX提取试剂盒适用于有限的样本（即，3毫米打孔量的提取可产生20次扩增）。GFX提取试剂盒为标准化干血滤纸片DNA提取提供了一种有用的方法。
