Cloning Full-Length Transcripts and Transcript Variants Using 5′ and 3′ RACE
Cloning Full-Length Transcripts and Transcript Variants Using 5′ and 3′ RACE
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DOI：
10.1007/978-1-60327-369-5_1
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发表时间：
2013-01-01
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通讯作者：
Freeman, Lita A.
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作者：
Freeman, Lita A.
必须克隆基因转录本和转录本变体以表征基因功能和调控。然而，获得从 5' 端到 3' 端具有准确序列的全长 cDNA 可能具有挑战性。在这里，我们描述了一种基于逆转录酶的方法，使用 Clontech 开发的 SMARTer（“RNA 末端的开关机制”）RACE 技术获得全长 cDNA。从感兴趣的组织中分离出 RNA，并与引物（用于聚 A+ 转录物的修饰寡 (dT) 引物；用于聚 A 转录物的随机六聚体或基因特异性引物）退火。修饰的MMLV逆转录酶使用引物从退火至引物的RNA转录物开始cDNA合成，并继续向转录物的5'端合成cDNA（逆转录）。重要的是，这种逆转录酶具有末端转移酶活性，因此当它到达转录物的 5' 末端时，它会在新合成的 cDNA 链上添加 3-5 个残基“尾部”。逆转录酶反应混合物中包含含有序列标签的寡核苷酸以及一系列末端修饰碱基，这些碱基与新合成的 cDNA 上的 3-5 个残基尾部退火。逆转录酶从转录物的末端开始进入修饰的碱基和序列标记寡核苷酸的其余部分。新合成的 cDNA 现在附有序列标签，可用作 PCR 模板，其中一个引物与序列标签互补，第二个引物对感兴趣的基因具有特异性。该片段可以被克隆并测序或者直接测序。如果使用高质量、未降解的 RNA，则可以大大提高获得转录本真实 5' 末端的能力。与 3' RACE 结合，可以轻松克隆全长转录本。该方法提供了重要调控区域（例如 5' 和 3' UTR 以及侧翼区域）的序列信息，非常适合检测转录物变体，包括具有替代转录起始位点、替代剪接和/或替代聚腺苷酸化的转录物变体。
Gene transcripts and transcript variants must be cloned to characterize gene function and regulation. However, obtaining full-length cDNAs with accurate sequences from the 5' end through to the 3' end can be challenging. Here we describe a reverse-transcriptase-based method for obtaining full-length cDNAs using the SMARTer ("Switching Mechanism At RNA Termini") RACE technology developed by Clontech. RNA is isolated from the tissue of interest and annealed to a primer (a modified oligo(dT) primer for polyA+ transcripts; random hexamers or a gene-specific primer for polyA-transcripts). A modi fied MMLV-reverse transcriptase uses the primer to initiate cDNA synthesis from RNA transcript(s) annealed to the primer and continues cDNA synthesis (reverse transcription) towards the 5' end of the transcript(s). Importantly, this reverse transcriptase possesses terminal transferase activity, so when it reaches the 5' end of a transcript it adds a 3-5 residue "tail" to the newly synthesized cDNA strand. Included in the reverse transcriptase reaction mix is an oligonucleotide containing a sequence tag as well as a terminal series of modi fied bases that anneal to the 3-5 residue tail on the newly synthesized cDNA. The reverse transcriptase proceeds from the end of the transcript onwards into the modi fied bases and the rest of the sequence-tagged oligo. The newly synthesized cDNA now has a sequence tag attached to it and can be used as a template for PCR, with one primer complementary to the sequence tag and the second primer specific to the gene of interest. The fragment can be cloned and sequenced or just sequenced directly. If high-quality, undegraded RNA is used, obtaining the true 5' end of a transcript is greatly enhanced. In combination with 3' RACE, full-length transcripts are easily cloned. This method provides sequence information on important regulatory regions, such as 5' and 3' UTRs and flanking regions, and is ideal for detecting transcript variants, including those with alternative transcriptional start sites, alternative splicing, and/or alternative polyadenylation.