Stable Isotope-Labeled ssDNA Biosynthesis via E. coli
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Solution Overview
Problem
Current commercial methods for obtaining stable isotope-labeled single-stranded DNA (ssDNA) are costly and inefficient, especially for sequences with high GC content, which often result in base pair mismatches and polymerase action hindrance due to secondary structure issues.
Innovation Solution
A method for preparing stable isotope-labeled ssDNA by biosynthesis with Escherichia coli (E. coli) using enzymatic digestion, which involves adding restriction endonuclease sites to the target sequence, ligating in tandem, and culturing in a medium with 15NH4Cl and/or 13C-glucose as sole nitrogen and carbon sources.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If solid-phase synthesis method is used to obtain stable isotope-labeled ssDNA, then the ssDNA can be produced, but the synthesis cost is extremely high (about 330,000 RMB for 1 mg of 24 nt ssDNA)
Solution Approach 1:
The patent uses inexpensive bacterial culture media and common laboratory reagents (restriction endonucleases, ligases, transformation reagents) instead of expensive solid-phase synthesis reagents. The E. coli cells serve as disposable factories that can be cultured cheaply and then discarded after DNA extraction, dramatically reducing the cost of producing isotope-labeled ssDNA
Solution Approach 2:
The E. coli bacterial system performs the DNA synthesis and isotope incorporation automatically through its natural replication machinery. The bacteria take up the recombinant plasmid and autonomously replicate it, incorporating 15N and/or 13C isotopes into the DNA during replication, eliminating the need for expensive chemical synthesis processes
2Manufacturing precision
If solid-phase synthesis method is used for DNA sequences with high GC content, then synthesis can be attempted, but base pair mismatches and polymerase action hindrance occur due to secondary structure formation
Solution Approach 1:
The patent replaces the mechanical/chemical solid-phase synthesis process with a biological system (E. coli replication machinery). The bacterial polymerase and replication system naturally handle high GC content sequences without the secondary structure problems that plague chemical synthesis, as the biological system has evolved mechanisms to process diverse DNA sequences efficiently
Solution Approach 2:
The patent changes the fundamental parameter of the synthesis system from chemical (solid-phase) to biological (in vivo replication). This parameter change allows the system to handle high GC content sequences effectively, as the biological replication machinery operates under different physical and chemical conditions that avoid the secondary structure formation issues encountered in chemical synthesis
3Productivity
If biosynthesis with E. coli is used to prepare stable isotope-labeled ssDNA, then the synthesis cost is reduced and efficiency is increased, but additional steps (cloning, transformation, digestion) are required
Solution Approach 1:
The patent merges multiple functions into the bacterial system: DNA replication, isotope incorporation, and DNA amplification all occur simultaneously within the E. coli cells during culture. The restriction digestion step then cleanly separates the target ssDNA from the rest of the bacterial DNA, providing both amplification and purification in an integrated workflow
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 significantly reduces the synthesis cost and increases the efficiency of in vitro synthesis of 15N and/or 13C-labeled ssDNA, while maintaining high labeling efficiency and yield.
Implementation Method 1
culturing in a medium with 15NH4Cl and/or 13C-glucose as sole nitrogen and carbon sources
Implementation Method 2
biosynthesis with Escherichia coli (E. coli)
Implementation Method 3
digesting the recombinant vector obtained in step 5) using the first restriction endonuclease and the second restriction endonuclease to obtain an asymmetric dsDNA
Implementation Method 4
the isolating is isolating by urea-polyacrylamide gel electrophoresis
Data Source
AI summary
Provided is a method for preparing stable isotope-labeled single-stranded DNA (ssDNA) by biosynthesis with E. coli, and the 15NH4Cl or 13C-Glcose is used as the only nitrogen or carbon source, which may significantly reduce costs. In the method of the present disclosure, the target sequence of ssDNA is tandemly duplicated on a high-copy vector, a site for a first restriction endonuclease and a site for a second restriction endonuclease are added to the 5′ and 3′ ends of the target sequence, respectively, and the recombinant vector is digested to obtain an asymmetric double-stranded DNA structure, which is then isolated by denaturation to obtain two ssDNAs of unequal lengths, including 15N- or 13C-labeled target ssDNA. The method of the present disclosure is able to effectively increase the yield of ssDNA, thereby improving the efficiency of in vitro synthesis of isotope labeled ssDNA.