Part:BBa_K4664006
Synthetic short cut
Exponential amplification of this synthetic short cut was observed in the one-pot assay, as opposed to linear amplification of the synthetic short cut in the two-step assay.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Background
Cas12a cis and trans cleavages cut the product of RCA amplification into segments with different lengths. These are then used in place of miRNA to anneal to padlocks and start secondary RCA processes. However, due to the linear ssDNA nature of padlocks, longer amplicons that have more complementary sequences could potentially form linear duplexes instead of the intended circularised duplexes. Thus, we hypothesised that amplicon segments shorter in length could better enhance secondary RCA kinetics.
Design
Sequence:
GGTCGAGCTGGACGGCGACGATCGATGGTTAGGGTATCTTAGCTTATCAGACTGATGTTGA
Results
Figure 1 shows the results of experiments with short-cut input. A paired T-test was carried out for the 100 fM short-cut vs. NC. P value is less than 0.0001, meaning statistically significant difference. Data is shown in Table 1.
Table 1. 100 fM short-cut vs. NC.
Figure 1. Synthetic short-cuts.
References
Jin, J., Vaud, S., Zhelkovsky, A., Pósfai, J., & McReynolds, L. A. (2016). Sensitive and specific miRNA detection method using SplintR Ligase. Nucleic Acids Research, 44(13), e116. https://doi.org/10.1093/nar/gkw399
He, Y., Wen, Y., Tian, Z., Hart, N. T., Han, S., Hughes, S. J., & Zeng, Y. (2023b). A one-pot isothermal Cas12-based assay for the sensitive detection of microRNAs. Nature Biomedical Engineering. https://doi.org/10.1038/s41551-023-01033-1
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