Part:BBa_K4636029
RCA_indu_1802_2
Rolling circle amplification (RCA) can generate long-repeated complementary DNA (cDNA) by elongation of primer. We design primer that is complementary to circDNA that can enhance specificity.
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]
In a typical rolling circle amplification (RCA) reaction, phi29 DNA Polymerase is commonly employed with random primers to conduct RCA, achieving isothermal amplification of circular DNA. However, in our project, we utilize a reverse transcriptase enzyme for conducting an RCA reaction, enabling the isothermal amplification of circular RNA (circRNA) and the generation of single-stranded complementary DNA for subsequent analysis. (Figure 1) Given that our RCA target is circRNA, to enhance the RCA reaction's specificity for circRNA, we not only design primers for specific RNA sites but also design primers target 5' and 3' termini of the RNA, known as back-splicing sites.[1]
Figure 1. Schematic diagram of RCA process. In the reaction, circRNA will be reverse transcribed by Induro® Reverse Transcriptase (New England Biolabs), generating long-repeated ssDNA.
After conducting literature reviews, we found that the majority of researchers use random primers, and no guidelines were found for designing RCA primers. Our secondary PI mentioned that designing primers independently can enhance specificity. Besides, if the Tm value of primer is close to our enzyme’s reaction temperature, we don’t need to anneal our primer with sample at lower temperature first. Primer design primarily involves using SnapGene software[2] to manually search for primers on the complementary insert sequence (BBa_K46360040 & BBa_K46360041) that have Tm values close to the enzyme’s reaction temperature and GC content around 50%. These primers are then individually tested using the IDT OligoAnalyzer™ Tool[3], with any primers prone to self-dimer formation being discarded. Finally, the remaining primer candidates are tested against each other using the IDT OligoAnalyzer™ Tool to assess the possibility of forming hetero primer.
Rolling circle amplification (RCA) can generate long-repeated complementary DNA (cDNA) by elongation of primer. We conduct RCA using two primers (RCA_indu_1802_1 and RCA_indu_1802_2) at 60 ℃. As can be seen in Figure 2, lane 2 shows a higher position than lane 4, indicating the successful generation of long-repeated complementary DNA (cDNA) by our primer.
Figure 2. Gel electrophoresis results verify the RCA process. Lane 1: 1000 bp ssDNA ladder. Lane 2: Circular RNA (202 ng) after 15 minutes RCA amplification. Lane 3: Circular RNA (202 ng) after 30 minutes RCA amplification. Lane 4: Low concentration of linear RNA (202 ng) after RCA 15 minutes amplification. Lane 5: Low concentration of linear RNA (202 ng) after RCA 30 minutes amplification. Lane 6: High concentration of linear RNA (2457.2 ng) after RCA 15 minutes amplification. Lane 7: High concentration of linear RNA (2457.2 ng) after RCA 30 minutes amplification.
1. Boss, M., & Arenz, C. (2020). A Fast and Easy Method for Specific Detection of Circular RNA by Rolling-Circle Amplification. *Chembiochem : a European journal of chemical biology*, *21*(6), 793–796. https://doi.org/10.1002/cbic.201900514
2. https://www.snapgene.com/
3. https://sg.idtdna.com/pages/tools/oligoanalyzer
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