采用3' cDNA末端快速扩增技术绘制癌症中的转录本图谱。
Adapting 3' Rapid Amplification of CDNA Ends to Map Transcripts in Cancer.
作者信息
Masamha Chioniso Patience, Todd Zachery
机构信息
Department of Pharmaceutical Sciences, College of Pharmacy and Health Sciences, Butler University;
Department of Pharmaceutical Sciences, College of Pharmacy and Health Sciences, Butler University.
出版信息
J Vis Exp. 2018 Mar 28(133):57318. doi: 10.3791/57318.
Maturation of eukaryotic mRNAs involves 3' end formation, which involves the addition of a poly(A) tail. In order to map the 3' end of a gene, the traditional method of choice is 3' rapid amplification of cDNA ends (3' RACE). Protocols for 3' RACE require the careful design and selection of nested primers within the 3' untranslated region (3' UTR) of the target gene of interest. However, with a few modifications the protocol can be used to include the entire 3' UTR and sequences within the open reading frame (ORF), providing a more comprehensive picture of the relationship between the ORF and the 3' UTR. This is in addition to identification of the polyadenylation signal (PAS), as well as the cleavage and polyadenylation site provided by conventional 3' RACE. Expanded 3' RACE can detect unusual 3' UTRs, including gene fusions within the 3' UTR, and the sequence information can be used to predict potential miRNA binding sites as well as AU rich destabilizing elements that may affect the stability of the transcript.
真核生物mRNA的成熟涉及3'端的形成，这包括添加一个聚腺苷酸（poly(A)）尾巴。为了绘制基因的3'端图谱，传统的首选方法是3' cDNA末端快速扩增（3' RACE）。3' RACE方案需要在目标基因的3'非翻译区（3' UTR）内仔细设计和选择巢式引物。然而，经过一些修改，该方案可用于包含整个3' UTR以及开放阅读框（ORF）内的序列，从而更全面地了解ORF与3' UTR之间的关系。这除了能够鉴定聚腺苷酸化信号（PAS）以及传统3' RACE所提供的切割和聚腺苷酸化位点之外。扩展的3' RACE可以检测异常的3' UTR，包括3' UTR内的基因融合，并且该序列信息可用于预测潜在的miRNA结合位点以及可能影响转录本稳定性的富含AU的不稳定元件。
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