Blocking PCR Amplification for Controlled DNA Repeat Synthesis
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current methods for synthesizing moderately and highly repetitive DNA sequences in vitro are limited by high costs, complex operation steps, and high error rates, making them unsuitable for industrial applications.
Innovation Solution
The method employs blocking PCR-based repeat expansion using three complementarily paired DNA primers and Taq DNA polymerase, allowing for controlled amplification of repetitive DNA sequences through cyclic heating and cooling, reducing the need for biologically active enzymes and simplifying the synthesis process.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If RCA method is used for synthesizing repetitive DNA sequences, then the synthesis can be achieved through cyclization amplification, but the operation steps become cumbersome and the cost of biologically active enzymes increases
Solution Approach 1:
The invention extracts and eliminates the cyclization ligation step from the RCA process by using a linear template with terminal repeat sequences that directly serve as priming sites. This removes the need for ligase and the complex cyclization operation, simplifying the workflow while maintaining amplification capability
Solution Approach 2:
The terminal repeat sequences on the linear template perform multiple functions: they serve as both the template for amplification and as the priming sites for DNA polymerase extension. This multi-functionality eliminates the need for separate primers and cyclization steps, reducing operational complexity
2Reliability
If RCA method is used for synthesizing repetitive DNA sequences, then amplification can be achieved, but the copy number control becomes difficult and the product length becomes unpredictable
Solution Approach 1:
The template is pre-designed with a defined number of terminal repeat sequences at its ends before amplification begins. This preliminary structuring ensures that the copy number in the final product is predetermined by the template design rather than being uncontrollably determined during amplification, enabling precise manufacturing control
Solution Approach 2:
The amplification process creates multiple copies of the terminal repeat sequences from the linear template. By controlling the number of repeats in the original template, the method ensures that each amplified product contains a predictable and controllable number of repeat units, achieving precise copy number control
3Reliability
If Gene SOE method is used for synthesizing highly repetitive DNA sequences, then gene assembly can be achieved, but base pairing slippage and mismatch occur leading to decreased splicing efficiency and increased error rate
Solution Approach 1:
The invention replaces the ligase-based mechanical joining system of Gene SOE with a DNA polymerase-based enzymatic synthesis system. This substitution eliminates the need for base pairing slippage and ligation steps that cause errors, using instead a more accurate polymerase extension mechanism that maintains high manufacturing precision even with highly repetitive sequences
4Reliability
If Gene SOE method is used for synthesizing highly repetitive DNA sequences, then gene assembly can be achieved, but the synthesis period becomes longer
Solution Approach 1:
The amplification process operates continuously through multiple cycles of denaturation, annealing, and extension without interruption. The DNA polymerase continuously extends the primers across all terminal repeat sequences in each cycle, maximizing the useful action time and significantly reducing the overall synthesis period compared to stepwise assembly methods
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This approach enables rapid, cost-effective, and precise synthesis of repetitive DNA sequences with controlled copy numbers, suitable for industrial-scale production and applications in biotechnology and material science.
Implementation Method 1
Blocking-type chain polymerization amplification reaction based in vitro fast synthesis of medium and high-copy DNA repetitive sequence
Implementation Method 2
cyclic heating and cooling
Data Source
AI summary
The invention provides a method for synthesizing a DNA sequence comprising repeat units, including designing and synthesizing an extension primer and a blocking primer based on the repeat unit, performing a PCR amplification reaction by using the repeat unit (as an amplification template), the extension primer, and the blocking primer in a PCR reaction system, to obtain the DNA sequence comprising repeat units. The invention also provides a kit for this method. The method of the invention has the characteristics such as controllable copy number for repeat synthesis, simple synthesis steps, and low cost, and is very suitable for high-throughput production in industry.