Part:BBa_K4308003
Padlock probe 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.
Padlock probe is a long single-stranded oligonucleotide fragment with two ends complementary to the target sequence and a junction sequence in the middle. It can be used as a binding site for universal primers. Padlock probe is involved in composing the RCA system. This system has been used to prepare DNA nanosponge.
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 nucleic acid amplification technology established by referring to the rolling circle replication of DNA molecules of circular pathogenic microorganisms in environment. It is a DNA amplification technology that occurs at a constant temperature. In the RCA reaction, when there is a circular DNA template and polymerase, by the action of DNA polymerase, the circular DNA is used as the template for replication, and finally a single strand DNA is formed, with a repeat sequence complementary to the circular DNA template.
Usage
Padlock probe is an important part of the RCA system. RCA system was used to prepare DNA nanosponge.
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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