Coutlée F, Bobo L, Mayur K, Yolken R H, Viscidi R P
Eudowood Division of Infectious Diseases, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
Anal Biochem. 1989 Aug 15;181(1):96-105. doi: 10.1016/0003-2697(89)90399-0.
A quantitative, nonisotopic hybridization assay which measures specific DNA-RNA hybrids is described. A biotinylated RNA probe is reacted in solution with a DNA target and the labeled hybrids are immobilized onto a solid phase surface with an antibiotin antibody. Bound hybrids are detected with a beta-galactosidase-labeled monoclonal antibody against DNA-RNA hybrids and are quantitated with the addition of a fluorogenic substrate. In a model system using pSP65 or pGEM4 plasmids and transcripts, biotinylated RNA probes allowed detection of 5 pg of DNA in 10(6) pg of exogenous nucleic acids in 1000 min. Signals generated in the system depended on input target length. A nucleic acid target of 25 bases was still detectable in the assay. Human immunodeficiency virus type 1 (HIV-1) DNA was amplified in the polymerase chain reaction with Taq polymerase and a set of primers for the pol gene, one of which contained T7 RNA polymerase promoter sequences. A HIV-RNA probe of 326 bases was transcribed with T7 RNA polymerase using polymerase chain reaction (PCR) amplified DNA as a template. The RNA probe of 326 bases performed as well as a RNA probe of 2588 bases for detection of a DNA segment of 355 bp. For detection of dilutions of HIV-1 with PCR, a set of primers (outer set) was used for amplification of HIV-1 DNA. In a separate reaction a set of primers nested between the first set generated through PCR an amplified DNA fragment with the T7 promoter. This fragment was transcribed for the synthesis of a biotinylated RNA probe. This probe could then be reacted with material amplified with the outer set of primers. Ten copies of HIV-DNA could be detected with this procedure.
本文描述了一种用于测量特定DNA-RNA杂交体的定量、非同位素杂交检测方法。生物素化的RNA探针在溶液中与DNA靶标反应，标记的杂交体通过抗生物素抗体固定在固相表面。用针对DNA-RNA杂交体的β-半乳糖苷酶标记的单克隆抗体检测结合的杂交体，并通过添加荧光底物进行定量。在使用pSP65或pGEM4质粒及转录本的模型系统中，生物素化的RNA探针在1000分钟内可检测出10⁶ pg外源核酸中5 pg的DNA。系统中产生的信号取决于输入靶标的长度。在该检测中，25个碱基的核酸靶标仍可检测到。人免疫缺陷病毒1型（HIV-1）DNA在聚合酶链反应中用Taq聚合酶和一组针对pol基因的引物进行扩增，其中一个引物含有T7 RNA聚合酶启动子序列。以聚合酶链反应（PCR）扩增的DNA为模板，用T7 RNA聚合酶转录出326个碱基的HIV-RNA探针。对于检测355 bp的DNA片段，326个碱基的RNA探针与2588个碱基的RNA探针表现相当。为了检测PCR扩增的HIV-1稀释液，使用一组引物（外部引物组）扩增HIV-1 DNA。在另一个反应中，一组嵌套在通过PCR产生的第一组引物之间的引物生成了带有T7启动子的扩增DNA片段。该片段被转录以合成生物素化的RNA探针。然后该探针可与用外部引物组扩增的材料反应。用该方法可检测到10份HIV-DNA拷贝。
