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. Author manuscript; available in PMC: 2023 Jul 25.
Published in final edited form as: Nat Biomed Eng. 2022 Aug 11;6(8):944–956. doi: 10.1038/s41551-022-00917-y

Fig. 3 |. rLAMP for two layers of nucleic acid amplification.

Fig. 3 |

a, Schematic of rLAMP mechanism for exponential DNA amplification using F3/B3 and FIP/BIP primers, resulting in higher-order inverted repeat structures. Red arrows indicate location of T7 promoter sequence, inserted in the mBIP primer. Upon T7 transcription, the resulting RNA contains one or more copies of the Cas13 target sequence (orange). b, Schematic of the location of different T7 promoter locations on the rLAMP dumbbell structure and loop primers. c, rLAMP time to threshold of six distinct T7 promoter insertions. Replicates that did not amplify are represented at 0 min. Error bars represent mean ± s.d. of amplified technical replicates, n = 4. d, Denaturing PAGE gels of mBIP rLAMP products to verify T7-mediated transcription. AfeI cleaves in the crRNA target region of templated products, which is expected to result in a single major transcribed species. e, Kinetics of T7 transcription and Cas13 detection on mBIP rLAMP products. Values are mean ± s.d. with n = 3 technical replicates. f, Cas13 detection of eight technical replicates of mBIP rLAMP amplification on genomic RNA, depicted as fold change over NTC at different reaction endpoints.