Part:BBa_K4245131
Middle sequence
This part is the middle sequence used within Lambert iGEM’s padlock probes for Rolling Circle Amplification (RCA). The middle sequence was designed by researchers at Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province at Shaanxi Normal University and used within their padlock probe design (Zhou et al., 2015). The complement of this sequence within the Rolling Circle Product (RCP) served as the target for the linear probes 6-carboxyl-fluorescein (FAM) Labeled DNA Probe (BBa_K4245130) and Black Hole Quencher 1 (BHQ1) DNA Probe (BBa_K4245132), such that each half of the middle sequence complement in RCP was complementary to each linear probe where the binding of the linear probes to the RCP middle sequence would bring the fluorophore and the quencher in close proximity, allowing the fluorescence to be quenched by the quencher (Zhou et al., 2015).
Lambert iGEM also used the split Lettuce DNA fluorescent aptamer developed by the Jaffrey Lab at the Weill-Cornell Medical College— Lettuce left (BBa_K4245134) and Lettuce right (BBa_K4245135)— so that the attachment of each component to each corresponding half of the middle sequence complement within the RCP would bring the split Lettuce together, thus allowing it to induce fluorescence of a GFP fluorophore mimic (Z)-4-(3,5-difluoro-4-hydroxybenzylidene)-2-methyl-1-(2,2,2-trifluoroethyl)-1H-imidazol-5(4 H)-one (DFHBI-1T) (VarnBuhler et al., 2022).
References
Zhou, F., Li, B., & Ma, J. (2015). A linear DNA probe as an alternative to a molecular beacon for improving the sensitivity of a homogenous fluorescence biosensing platform for DNA detection using target-primed rolling circle amplification. RSC Advances, 5(6), 4019–4025. https://doi.org/10.1039/c4ra14467h
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]
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