Green D A, Deutsch W A
Anal Biochem. 1984 Nov 1;142(2):497-503. doi: 10.1016/0003-2697(84)90495-0.
A simple but effective technique for determining the presence of uracil existing as either A:U base pairs or G:U base pairs in DNA was developed. DNA is degraded to deoxynucleoside 3'-monophosphates by a combination of micrococcal nuclease and spleen phosphodiesterase. The monophosphates are converted to 5'-end-labeled 32P-labeled diphosphates in a reaction catalyzed by T4 polynucleotide kinase. The resultant product is then converted to 5'-end-labeled deoxynucleoside monophosphates by P1 nuclease digestion, which specifically removes 3'-phosphates. Successful separation of labeled dUMP from conventional bases in DNA is achieved by two-dimensional polyethyleneimine chromatography, with its detection determined by autoradiography and liquid scintillation counting. The sensitivity of the technique described can detect a minimum 1 X 10(-16) mol of dUMP in DNA. Additionally, the detection of 5-methylcytosine in placental DNA demonstrates the flexibility of the technique for the analysis of modified bases in DNA.
开发了一种简单而有效的技术，用于确定DNA中以A:U碱基对或G:U碱基对形式存在的尿嘧啶。通过微球菌核酸酶和脾磷酸二酯酶的联合作用，将DNA降解为脱氧核苷3'-单磷酸。在T4多核苷酸激酶催化的反应中，单磷酸转化为5'-末端标记的32P标记的二磷酸。然后通过P1核酸酶消化将所得产物转化为5'-末端标记的脱氧核苷单磷酸，P1核酸酶可特异性去除3'-磷酸。通过二维聚乙烯亚胺色谱法成功地从DNA中的常规碱基中分离出标记的dUMP，通过放射自显影和液体闪烁计数来确定其检测结果。所描述技术的灵敏度可检测到DNA中最低1×10(-16)摩尔的dUMP。此外，对胎盘DNA中5-甲基胞嘧啶的检测证明了该技术在分析DNA中修饰碱基方面的灵活性。
