Part:BBa_K4636038
AELA_PCR_fw
The Asymmetric Exponential and Linear Amplification (AELA) represents a novel variant of PCR capable of producing single-stranded amplicons from double-stranded DNA. According to reference paper[1], AELA-PCR utilizes a unique amplification strategy that combines both exponential and linear amplification of the target, requiring the use of specially designed primers.
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]
Design
The primer design for AELA-PCR is divided into two parts: the extended primer and the unextended primer, as shown in Figure 1. Here, we will discuss considerations for both.
Figure 1. AELA-PCR primer design comprises both the extended primer and the unextended primer.
Extended primer (rv):
1. Primer size between 25~30 bps.
2. Tm value between 68~70°C.
3. We should design self-complementary sequence for AELA PCR linear amplification phase.
4. Value of GC content should be 40%~60%.
5. 3’ end with high GC content.
Unextended primer (fw):
(Design based on standard PCR primer design)[2]
1. Primer size between 18~24 bps.
2. Tm value between 50~60°C.
3. Value of GC content should be 40%~60%.
4. 3’ end with high GC content.
We used Primer 3[3] to search the suitable site an check it by IDT OligoAnalyzer™ Tool.[4]
Experiment
We failed in the previous step. (Colony PCR)
Reference
[1] Reddy Banda, S., Klapproth, H., Smit, N., Bednar, S., Brandstetter, T., & Rühe, J. (2022). An advanced and efficient asymmetric PCR method for microarray applications. Frontiers in bioengineering and biotechnology, 10, 1045154. https://doi.org/10.3389/fbioe.2022.1045154
[2] https://www.addgene.org/protocols/primer-design/
[3] https://primer3.ut.ee/
[4] https://sg.idtdna.com/pages/tools/oligoanalyzer
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