Lee L G, Connell C R, Bloch W
Applied Biosystems, Division of Perkin-Elmer, Foster City, CA 94404.
Nucleic Acids Res. 1993 Aug 11;21(16):3761-6. doi: 10.1093/nar/21.16.3761.
Nick-translation PCR was performed with fluorogenic probes. Two probes were used: one complementary to a sequence containing the F508 codon of the normal human cystic fibrosis (CF) gene (wt DNA) and one complementary to a sequence containing the delta F508 three base pair deletion (mut DNA). Each probe contained a unique and spectrally resolvable fluorescent indicator dye at the 5' end and a common quencher dye attached to the seventh nucleotide from the 5' end. The F508/delta F508 site was located between the indicator and quencher. The probes were added at the start of a PCR containing mut DNA, wt DNA or heterozygous DNA and were degraded during thermal cycling. Although both probes were degraded, each probe generated fluorescence from its indicator dye only when the sequence between the indicator and quencher dyes was perfectly complementary to target. The identify of the target DNA could be determined from the post-PCR fluorescence emission spectrum.
采用荧光探针进行缺口平移聚合酶链反应（Nick-translation PCR）。使用了两种探针：一种与正常人囊性纤维化（CF）基因的包含F508密码子的序列互补（野生型DNA），另一种与包含ΔF508三个碱基对缺失的序列互补（突变型DNA）。每个探针在5'端含有一种独特且光谱可分辨的荧光指示剂染料，并且在距5'端第七个核苷酸处连接有一个通用淬灭剂染料。F508/ΔF508位点位于指示剂和淬灭剂之间。将探针添加到包含突变型DNA、野生型DNA或杂合DNA的PCR起始反应中，并在热循环过程中降解。尽管两种探针都被降解，但每个探针仅在指示剂和淬灭剂染料之间的序列与靶标完全互补时才从其指示剂染料产生荧光。可以从PCR后的荧光发射光谱确定靶标DNA的身份。
