Nucleic Acids Research介绍新实时定量PCR技术

　　

生物通报道：近日，研究人员开发了一个新型实时定量PCR技术，该技术诱使PCR产物折叠成为哑铃状，并通过FRET探针实时跟踪PCR的进行。文章发表在Nucleic Acids Research杂志上。

Igor Kutyavin将自己的这个新技术称为Angler PCR，该技术所用的荧光探针只含有几个核苷酸。这么短的探针意味着，可以创建包含所有可能的探针文库，在不牺牲质量的情况下，大幅削减实时定量PCR的成本。

Angler PCR利用PCR引物，给扩增子两端添上额外的核苷酸，使两个末端形成环状，折叠成为哑铃形。在哑铃的两个环之间，还留有一段短核苷酸序列，允许FRET（荧光共振能量转移）探针与之结合。当探针结合上来以后，Taq酶的外切活性会切除FRET探针上的荧光基团，此时游离的荧光基团发出荧光信号。

“在这种情况下，扩增与检测密不可分，因为扩增为检测提供了目标，”Kutyavin解释道。

为了检验这一技术，Kutyavin对96个随机供体的血液样本进行分析，希望通过Angler PCR鉴定各人基因组中的10个SNP。他仅用四个核苷酸组成的FRET探针，就成功鉴定了上述SNP。研究显示，Angler PCR获得了PCR产物的实时荧光曲线，其检测结果与传统Taqman法相差不多。

“与传统方法相比，Angler PCR有一个明显的优势，其探针不需要那么稳定，也不用每次实验都从头制备新探针，”Kutyavin说。传统PCR要求探针比引物更加稳定，而Angler PCR的探针则没有这样的限制。

这是因为，Angler PCR探针只需要四个核苷酸。Kutyavin指出，可以构建PCR探针文库，只需256个探针就能覆盖所有可能的组合。这样的文库，有望大大降低制备探针的成本，从而实现更经济简便的实时定量PCR。

不过，目前Angler PCR还难以推广应用，因为还缺乏相应的探针设计或数据分析软件。“目前我们只能亲自完成这些工作，”Kutyavin说。他希望尽快将这一技术商业化，并找到合作伙伴共同开发合适的应用软件。

 

（生物通编辑：叶予）

生物通推荐原文摘要：

Use of extremely short Forster resonance energy transfer probes in real-time polymerase chain reaction

Described in the article is a new approach for the sequence-specific detection of nucleic acids in real-time polymerase chain reaction (PCR) using fluorescently labeled oligonucleotide probes. The method is based on the production of PCR amplicons, which fold into dumbbell-like secondary structures carrying a specially designed 'probe-luring' sequence at their 5' ends. Hybridization of this sequence to a complementary 'anchoring' tail introduced at the 3' end of a fluorescent probe enables the probe to bind to its target during PCR, and the subsequent probe cleavage results in the florescence signal. As it has been shown in the study, this amplicon-endorsed and guided formation of the probe-target duplex allows the use of extremely short oligonucleotide probes, up to tetranucleotides in length. In particular, the short length of the fluorescent probes makes possible the development of a 'universal' probe inventory that is relatively small in size but represents all possible sequence variations. The unparalleled cost-effectiveness of the inventory approach is discussed. Despite the short length of the probes, this new method, named Angler real-time PCR, remains highly sequence specific, and the results of the study indicate that it can be effectively used for quantitative PCR and the detection of polymorphic variations.