Part:BBa_K4308002
Primer for RCA
Rolling circle amplification (RCA) is a rapid, sensitive and isothermal ssDNA amplification technique. The key to the occurrence of the RCA reaction is the construction of a complete single-stranded DNA loop for subsequent amplification, and the RCA looping mode includes sticky end looping and flat end looping. The RCA primer is able to bind to the padlock probe of RCA , triggering the RCA reaction.
We designed a nucleic acid nanosponge prepared by RCA to encapsulate the follow-up protein, and this sequence is the primer required for RCA. For circular template, add this single primer complementary to the template, and after hybridization between the primer and the template, roll loop amplification can be started. The synthetic product is repetitive linear single stranded DNA sequence, and the process is linear amplification.
Biology
RCA is a simple and efficient isothermal enzymatic amplification strategy for the synthesis of ultra-long single-stranded DNA that benefits from mild reaction conditions, as well as stability and efficiency in complex biological environments. Other available methods for DNA hydrogel synthesis include hybrid chain reactions that require a large number of hairpin strands to produce DNA strands and PCR that requires temperature cycling. In contrast, the RCA process is performed at constant temperature and requires a small amount of circular DNA template. It is the Primer of RCA that binds to Phi29 polymerase and initiates the RCA reaction.
Usage
The Primer of RCA was used to assist in initiating the RCA reaction.
Reaction system: 1×RCA Reaction Buffer, 10 U Phi29 Polymerase, 160 μM SAM，4 U M.SssI，1 mM dNTPs，1μM DNA template, 1 μM primer, 3 U glucoamylase, 1 μg MBD-SA.
Preparation process: the above system was incubated at 37 ℃ for more than 6 h, when the flocs appear, centrifuge it in 10000 g for 1 min, and remove the supernatant. At last, use 100 μL ddH2O to clean it for 3 times.
Characterization
1. Identification
We used the method of chemical synthesis to obtained primer of RCA.
2. Expression
In order to verify that the RCA reaction we designed can amplify the bands we wanted to obtain, we performed agarose gel electrophoresis on the reaction products.
Figure. 1 Agarose gel electrophoresis of different products of the whole RCA process. Lane 1: Linear DNA; Lane 2 : DNA with stem-loop; Lane 3, Lane4, Lane5: DNA nanosponge.
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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