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Fig. 2 | BMC Genomics

Fig. 2

From: Improvements to the HITS-CLIP protocol eliminate widespread mispriming artifacts

Fig. 2

Identification of misprimed reads using a nested reverse transcription primer. a When the reverse transcription primer (blue) is flush with the 3′ adaptor, reads originating from 3′ adaptor (emerald) priming and mispriming (vermilion) are indistinguishable, as both are competent PCR templates. b However, when a nested reverse transcription primer (blue) is used along with a protected reverse PCR primer (bases with phosphorothioate bonds are shown in sky blue), only reads originating from 3′ adaptor (emerald) priming are valid templates for PCR amplification. Reverse transcription products derived from mispriming are not amplified in subsequent PCR steps, as they do not contain the final 3 bases of the reverse PCR primer. This 3-base mismatch prevents elongation of the primer by the polymerase, and the phosphorothioate bonds prevent the ‘chew-back’ of the primer by exonuclease activity of the polymerase

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