Next-generation sequencing-based 5′ rapid amplification of cDNA ends for alternative promoters

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• 作者：Bambarendage P.U. Perera ; Joomyeong Kim ; ^{jkim@lsu.edu" class="auth_mail" title="E-mail the corresponding author}
• 关键词：5&prime ; RACE ; NGS ; Alternative promoters ; Peg3
• 刊名：Analytical Biochemistry
• 出版年：2016
• 出版时间：1 February 2016
• 年：2016
• 卷：494
• 期：Complete
• 页码：82-84
• 全文大小：450 K

文摘

Mammalian genomes contain many unknown alternative first exons and promoters. Thus, we have modified the existing 5′RACE (5′ rapid amplification of cDNA ends) approach into a next-generation sequencing (NGS)-based new protocol that can identify these alternative promoters. This protocol has incorporated two main ideas: (i) 5′RACE starting from the known second exons of genes and (ii) NGS-based sequencing of the subsequent cDNA products. This protocol also provides a bioinformatics strategy that processes the sequence reads from NGS runs. This protocol has successfully identified several alternative promoters for an imprinted gene, PEG3. Overall, this NGS-based 5′RACE protocol is a sensitive and reliable method for detecting low-abundant transcripts and promoters.