One-Step DNA Cloning with Type IIs Enzymes
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Solution Overview
Problem
Traditional DNA cloning methods using restriction enzymes are time-consuming and unreliable, especially for complex sequences or large fragments, and often result in the incorporation of unwanted recombinase recognition sequences that interfere with construct function.
Innovation Solution
A one-step molecular cloning system utilizing typeIIs restriction enzymes, which produce compatible ends that can be joined by DNA ligase, allowing for the creation of a polynucleotide without recognition sequences for these enzymes, thereby controlling the sequence and avoiding the incorporation of restriction sites or recombinase sequences.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If traditional restriction enzymes are used for DNA cloning, then the method is simple and well-established, but the process is time-consuming and unreliable
Solution Approach 1:
The patent combines multiple cloning steps into a single reaction mixture containing type IIs restriction enzymes and DNA ligase. The type IIs enzymes generate compatible ends that can be directly ligated in the same reaction, eliminating the need for separate digestion and ligation steps, thereby reducing time and improving reliability
Solution Approach 2:
The patent utilizes type IIs restriction enzymes that recognize non-palindromic sequences and cut outside the recognition site, creating compatible ends with specific overhangs. This parameter change in enzyme specificity enables direct ligation without traditional restriction sites, accelerating the cloning process
2Reliability
If traditional restriction enzymes are used, then the method is straightforward, but unwanted recombinase recognition sequences are incorporated that interfere with construct function
Solution Approach 1:
The patent extracts and eliminates the harmful recombinase recognition sequences from the final construct. By using type IIs enzymes that cut outside their recognition sites and designing primers that avoid these sequences, the method removes the source of interference while maintaining cloning efficiency
Solution Approach 2:
The patent converts the potential harm of restriction site scarring into a benefit by using type IIs enzymes that leave no recognizable sequences in the final product. The compatible ends generated by these enzymes enable clean ligation without leaving interfering sequences, turning a potentially harmful process into a beneficial one
3Adaptability or versatility
If multiple cloning sites are used to accommodate diverse vectors, then versatility is improved, but device complexity increases
Solution Approach 1:
The patent creates a universal cloning system where a single reaction mixture with type IIs enzymes and ligase can handle multiple vector types and insert combinations. The compatible ends generated by type IIs enzymes provide universal compatibility without requiring multiple specialized cloning sites, reducing system complexity while maintaining versatility
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 method enables rapid and reliable cloning of polynucleotides into multiple vectors or a single specialized vector, eliminating the need for multiple cloning sites and reducing the risk of sequence interference, thus enhancing the efficiency and reliability of DNA manipulation.
Implementation Method 1
a first polynucleotide comprising a selectable marker and two sequences recognized by typeIIs restriction enzymes; a typeIIs restriction enzyme... so that the first polynucleotide is joined to the second polynucleotide
Implementation Method 2
a DNA ligase so that the first polynucleotide is joined to the second polynucleotide
Data Source
AI summary
Methods and kits for joining two or more polynucleotides to form a product polynucleotide are provided. A mixture contains a first polynucleotide comprising a selectable marker. The mixture further contains a second polynucleotide comprising a first typeIIs recognition sequence and a second typeIIs recognition sequence. The second polynucleotide is other than the first polynucleotide. The mixture further contains a first typeIIs restriction endonuclease that cleaves the first typeIIs recognition sequence to produce a first end, a second typeIIs restriction endonuclease that cleaves the second typeIIs recognition sequence to produce a second end, and a DNA ligase. The first end is not compatible with the second end. The combined actions of the enzymes in the mixture join the first polynucleotide to the second polynucleotide forming a product polynucleotide, which is obtained by transforming the mixture into a host cell.