Zhang D Y, Brandwein M, Hsuih T C, Li H
The Lillian, Henry M. Stratton-Hans Popper Department of Pathology, Department of Otolaryngology, Mount Sinai School of Medicine, New York, NY 10029, USA.
Gene. 1998 May 12;211(2):277-85. doi: 10.1016/s0378-1119(98)00113-9.
We describe a novel polymerase chain reaction (PCR)-based gene amplification method utilizing a circularizable oligodeoxyribonucleotide probe (C-probe). The C-probe contains two target complementary regions located at each terminus and an interposed generic PCR primer binding region. The hybridization of C-probe to a target brings two termini in direct apposition as the complementary regions of C-probe wind around the target to form a double helix. Subsequent ligation of the two termini results in a covalently linked C-probe that becomes 'locked on to' the target. The circular nature of the C-probe allows for the generation of a multimeric single-stranded DNA (ssDNA) via extension of the antisense primer by Taq DNA polymerase along the C-probe and displacement of downstream strand, analogous to 'rolling circle' replication of bacteriophage in vivo. This multimeric ssDNA then serves as a template for multiple sense primers to hybridize, extend, and displace downstream DNA, generating a large ramified (branching) DNA complex. Subsequent thermocycling denatures the dsDNA and initiates the next round of primer extension and ramification. This model results in significantly improved amplification kinetics (super-exponential) as compared to conventional PCR. Our results show that the C-probe was 1000 times more sensitive than the corresponding linear hemiprobes for detecting Epstein-Barr virus early RNA. The C-probe not only increases the power of amplification but also offers a means for decontaminating carryover amplicons. As the ligated C-probes possess no free termini, they are resistant to exonuclease digestion, whereas contaminated linear amplicons are susceptible to digestion. Treatment of the ligation reaction mixture with exonuclease prior to amplification eliminated the amplicon contaminant, which could also have been co-amplified with the same PCR primers; only the ligated C-probes were amplified. The combined advantages of the C-probe and thermocycling have a broad applicability for the detection of both DNA and RNA. Finally, we described a novel isothermal amplification method, ramification extension amplification, utilizing circular nature of C-probe and displacement activity of DNA polymerase.
我们描述了一种基于聚合酶链反应（PCR）的新型基因扩增方法，该方法利用了可环化的寡脱氧核糖核苷酸探针（C-探针）。C-探针在每个末端包含两个靶标互补区域以及一个插入的通用PCR引物结合区域。C-探针与靶标杂交会使两个末端直接并列，因为C-探针的互补区域围绕靶标缠绕形成双螺旋。随后两个末端的连接会产生一个共价连接的C-探针，该探针“锁定”在靶标上。C-探针的环状性质允许通过Taq DNA聚合酶沿着C-探针延伸反义引物并置换下游链来生成多聚体单链DNA（ssDNA），这类似于噬菌体在体内的“滚环”复制。然后，这种多聚体ssDNA作为多个正义引物杂交、延伸和置换下游DNA的模板，产生一个大的分支状DNA复合物。随后的热循环使双链DNA变性，并启动下一轮引物延伸和分支反应。与传统PCR相比，该模型导致扩增动力学显著改善（超指数）。我们的结果表明，在检测爱泼斯坦-巴尔病毒早期RNA时，C-探针的灵敏度比相应的线性半探针高1000倍。C-探针不仅提高了扩增能力，还提供了一种去除残留扩增子污染的方法。由于连接后的C-探针没有游离末端，它们对外切核酸酶消化具有抗性，而受污染的线性扩增子则易受消化。在扩增前用外切核酸酶处理连接反应混合物可消除扩增子污染物，这些污染物也可能与相同的PCR引物共同扩增；只有连接后的C-探针被扩增。C-探针和热循环的综合优势在DNA和RNA检测方面具有广泛的适用性。最后，我们描述了一种新型等温扩增方法，即分支延伸扩增，该方法利用了C-探针的环状性质和DNA聚合酶的置换活性。
