Rolling Circle Amplification (RCA) is an isothermal amplification technology developed based on the circular pathogenic microorganism DNA/RNA molecular rolling replication in nature. Using a small segment of cyclic oligonucleotides as a template and dNTPs/NTPs as raw materials, a long repetitive single-stranded DNA/RNA is produced by amplification under the action of DNA/RNA polymerase. The amplification methods include linear amplification and exponential amplification. RCA has been widely used in genomics, proteomics, molecular diagnosis, biosensors, drug development, and other fields.
Operating Principle of RCA
The template of RCA must be circular. If linear genes are amplified, padlock probes are required. Both ends of the padlock probe have complementary sequences with the target gene. The target gene is identified by the padlock probe and combined to form an incompletely closed cyclic oligonucleotide. The combination of the padlock probe and the target gene can form a more stable topology to ensure the stability of the connection product, and then a fully closed cyclic oligonucleotide is formed under the action of the ligase. If the template itself is a cyclic single-stranded oligonucleotide, this process is not required. The two most common amplification processes are described below.
- Linear amplification: the positive primer recognizes the pairing sequence of the circular template, and under the action of phi29 DNA polymerase, the repetitive linear single-stranded DNA sequence is synthesized. This single-stranded DNA contains hundreds or thousands of repetitive template complementary fragments.
- Exponential amplification: On the basis of linear amplification, a reverse primer is added. This primer can combine with a linear single-stranded DNA sequence. Under the function of phi29 DNA polymerase's strand displacement, it can form a multi-branch amplification reaction, making the product increase exponentially.
Important Components of RCA
- Padlock probe: It has a complementary sequence with the target gene and combines with the target gene to form an incomplete closed loop.
- Forward primer: Identify the pairing sequence of the circular template and conduct linear amplification of RCA.
- Reverse primer: Identify the linear single-stranded sequence of linear amplification product and conduct RCA exponential amplification.
- DNA ligase: connect the two ends of the incomplete lock ring to form a completely closed circular template.
- phi29 DNA polymerase: Thermostable DNA polymerase cloned from Bacillus subtilis phage phi29, characterized by strong strand displacement activity and continuous synthesis reaction.
Key Points of RCA
- The RCA detection template should be a single-stranded circular structure. If it is linear DNA, it should be cyclized.
- For circular RNA templates, RNA reverse transcription is not required.
- phi29 DNA polymerase can catalyze DNA polymerization at room temperature, so RCA can react at 30°C or room temperature.
- The amplification efficiency of linear amplification can reach 105 times, and that of exponential amplification can reach 109 times.
- RCA has high specificity and is suitable for SNP detection.
- The products obtained from the phosphorylation of RCA products can be directly used for sequencing.
Solutions of SBS Genetech
phi29 HT DNA polymerase is an updated version of phi29 DNA polymerase. In addition to the strong strand displacement and continuous synthesis (> 70kb) activity of phi29 DNA polymerase, phi29 HT DNA polymerase can continuously synthesize DNA at 42°C, while the activity of phi29 DNA polymerase is very low at this temperature. In addition, phi29 HT DNA Polymerase still has a strong 3 '- 5' exonuclease proofreading function and the fidelity of the synthesized DNA fragments is high. The exonuclease activity of this enzyme is strong, so the primer needs 3'- end thio-modification in the process of synthesis to reduce the cleavage effect of the exonuclease activity on the primer.
The high-temperature reaction characteristic of phi29 HT DNA Polymerase has the following advantages:
- In the next generation sequencing (NGS), the enzyme has stronger amplification activity for complex templates such as high GC content and palindrome structure, which makes the coverage of NGS more uniform and reduces the depth required for sequencing.
- High-temperature reaction conditions improve the synthesis of WGA products of genomic DNA and can be used for variable temperature amplification.
- The gap region in sequencing is reduced, which can improve the quality and integrity of the data from single-cell sequencing.
- Reduce non-specific amplification products.
- Improve the amplification performance and specificity of MDA/RCA and other experiments.
For single-stranded linear oligonucleotides, the most important part of the cyclization process is the design of the padlock probe. The two ends of the padlock probe are specifically complementary to the target gene sequence. When there is a mismatch, the probe connection cannot be completed. High-quality probes can ensure the high specificity of DNA/RNA amplification.
At SBS Genetech, we provide guidance on the design of the padlock probe and combines it with the unique primer synthesis platform of RCA to ensure the formation of a high-quality cyclic oligonucleotide template for the amplification reaction.
Besides, SBS Genetech is recognized as one of the global major leading industry players in Oligonucleotide Synthesis by third-party market researchers. For more details, please visit DNA Oligonucleotide Synthesis Market Industry Analysis 2022 To 2028 based on Sales, Revenue, Size, and Share.