Voss H, Schwager C, Wirkner U, Sproat B, Zimmermann J, Rosenthal A, Erfle H, Stegemann J, Ansorge W
European Molecular Biology Laboratory, Heidelberg, FRG.
Nucleic Acids Res. 1989 Apr 11;17(7):2517-27. doi: 10.1093/nar/17.7.2517.
In vitro amplification of genomic DNA and total RNA, as well as recombinant DNA, using one fluorescently labelled and one unlabelled primer during amplification, together with on-line analysis of the products on the EMBL fluorescent DNA sequencer, is described. Further is reported direct sequencing of fluorescently labelled amplified probes by solid-phase chemical degradation, without subcloning and purification steps involved. At present up to 350 bases in 4 hours are determined with this technique. The fluorescent dye and its bond to the oligonucleotide are stable during the amplification cycles, and do not interfere with the enzymatic polymerization. High sensitivity of the detection device, down to 10(-18) moles, corresponding to less than 10(6) molecules makes possible analyses of the non-radioactive amplified probes after only 10 amplification cycles, starting with about 5 x 10(4) copies of recombinant DNA.
描述了在体外扩增基因组DNA、总RNA以及重组DNA的方法，即在扩增过程中使用一个荧光标记引物和一个未标记引物，并在EMBL荧光DNA测序仪上对产物进行在线分析。还报道了通过固相化学降解对荧光标记的扩增探针进行直接测序，无需进行亚克隆和纯化步骤。目前，用该技术在4小时内可测定多达350个碱基。荧光染料及其与寡核苷酸的结合在扩增循环中稳定，且不干扰酶促聚合反应。检测装置的高灵敏度可达10^(-18)摩尔，相当于少于10^6个分子，这使得仅从约5×10^4个重组DNA拷贝开始，经过10个扩增循环后就能对非放射性扩增探针进行分析。
