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Figure 10 | Mobile DNA

Figure 10

From: Biotechnological applications of mobile group II introns and their reverse transcriptases: gene targeting, RNA-seq, and non-coding RNA analysis

Figure 10

Thermostable group II intron reverse transcriptase (RT) template switching method for attachment of adaptor sequences for RNA-seq. The RT binds to an initial substrate consisting of a synthetic RNA oligonucleotide adaptor and an annealed DNA primer, with the RNA oligonucleotide having a blocking group attached to its 3’ end to impede recopying by the RT. In the example shown, the initial substrate has a single nucleotide 3’ overhang of the DNA primer (denoted N to signify that this nucleotide can be A, C, G, or T or a mixture of all four nucleotides). The single nucleotide 3’ overhang can facilitate template switching by base pairing to a complementary 3’ terminal nucleotide of a target RNA, which is a miRNA in the example shown. After template switching, the RT copies the target miRNA. The resulting cDNA containing the adaptor sequence seamlessly linked to the miRNA sequence is circularized with CircLigase and amplified by PCR with Primers P1 and P2 that introduce primer-binding sites and barcodes for RNA-seq. Relinearization of the circularized cDNA, which can increase the efficiency of the PCR in some cases, is an optional step [10].

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