一种将半抗原引入DNA探针的化学方法。

A chemical method for introducing haptens onto DNA probes.

作者信息

Keller G H, Cumming C U, Huang D P, Manak M M, Ting R

机构信息

Biotech Research Laboratories, Inc., Rockville, Maryland 20850.

出版信息

Anal Biochem. 1988 May 1;170(2):441-50. doi: 10.1016/0003-2697(88)90656-2.

DOI:10.1016/0003-2697(88)90656-2

PMID:3394942

Abstract

We have developed a versatile chemical method of attaching hapten moieties onto DNA, for the construction of nonisotopic DNA probes. The DNA is reacted with N-bromosuccinimide at alkaline pH, resulting in bromination of a fraction of the thymine, guanine, and cytosine residues, with adenine modified to a lesser extent. The bromine is subsequently displaced by a primary amino group, attached to a linker arm. The other end of the linker arm has a detectable group preattached to it. We have labeled cloned hepatitis B viral (HBV) DNA with the hapten 2,4-dinitrophenyl (DNP) and used it in combination with a high affinity rabbit anti-DNP antibody, for the detection of hepatitis B DNA by slot blotting. This probe was sensitive enough to specifically detect 1 X 10(-17) mol (1 X 10(6) copies) of HBV DNA in total DNA from human serum.

摘要

我们已经开发出一种通用的化学方法，用于将半抗原部分连接到DNA上，以构建非同位素DNA探针。DNA在碱性pH条件下与N-溴代琥珀酰亚胺反应，导致一部分胸腺嘧啶、鸟嘌呤和胞嘧啶残基发生溴化，腺嘌呤的修饰程度较小。随后，溴被连接到连接臂上的伯氨基取代。连接臂的另一端预先连接有可检测基团。我们用半抗原2,4-二硝基苯基（DNP）标记了克隆的乙型肝炎病毒（HBV）DNA，并将其与高亲和力的兔抗DNP抗体结合使用，通过狭缝印迹法检测乙型肝炎DNA。该探针灵敏度足以特异性检测人血清总DNA中1×10⁻¹⁷摩尔（1×10⁶个拷贝）的HBV DNA。