Nucleic Acid Isolation via Enzymatic Biotinylation and Affinity Binding
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Solution Overview
Problem
Current nucleic acid extraction methods from biological samples are inefficient and often result in non-specific binding, leading to loss of nucleic acids during purification, especially when using beads and columns, which rely on electric charge interactions and require multiple wash steps, resulting in incomplete recovery of nucleic acids.
Innovation Solution
A method involving direct enzymatic tagging of nucleic acids with polymeric tails containing affinity tags, allowing specific binding to solid supports coated with corresponding binding partners, enabling efficient isolation and purification without prior extraction or purification steps, using enzymes like terminal transferase to add biotinylated dNTPs or ddNTPs to the 3' ends of nucleic acids.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If beads and columns relying on electric charge interactions are used for nucleic acid purification, then nucleic acids can be captured from biological samples, but non-specific binding occurs leading to loss of nucleic acids during purification
Solution Approach 1:
The patent applies preliminary action by enzymatically tagging nucleic acids with affinity tags (such as biotin) before the purification step. This tagging is performed using terminal transferase to add modified dNTPs to the 3' ends of nucleic acids, ensuring that the nucleic acids are pre-marked for specific binding to the solid support, thereby avoiding non-specific binding losses during purification
Solution Approach 2:
The patent uses an intermediary affinity tag (such as biotin) that mediates the binding between nucleic acids and the solid support (streptavidin-coated beads). This intermediary enables highly specific binding through the biotin-streptavidin interaction, eliminating the non-specific charge-based binding problems of conventional methods
2Reliability
If multiple wash steps are performed during purification, then contaminants are removed, but nucleic acid recovery is incomplete due to loss during washing
Solution Approach 1:
The affinity tagging is performed in advance before purification, allowing the nucleic acids to be specifically marked and bound to the solid support. This preliminary tagging enables the nucleic acids to withstand multiple wash steps without loss, as they are firmly attached through the high-affinity biotin-streptavidin interaction while contaminants are removed
Solution Approach 2:
The biotin-streptavidin intermediary complex provides extremely strong and specific binding that can endure multiple wash steps. This intermediary system allows thorough purification to remove contaminants while maintaining complete nucleic acid recovery, as the affinity-bound nucleic acids do not detach during washing
3Reliability
If conventional extraction and purification steps are performed before isolation, then nucleic acids are prepared for binding, but the process becomes complex and time-consuming
Solution Approach 1:
The patent merges the tagging and purification steps into a single integrated process. The enzymatic tagging with affinity tags is performed directly in the biological sample without prior extraction, and the same reaction mixture contains the solid support for immediate binding. This consolidation eliminates separate extraction and purification steps, reducing complexity while maintaining nucleic acid quality
Solution Approach 2:
The reaction mix serves multiple functions: it contains the terminal transferase for tagging, the modified dNTPs for affinity tag incorporation, the buffer for enzymatic activity, and the solid support for binding. This multi-functional system eliminates the need for separate extraction and purification reagents and steps, simplifying the overall process
4Reliability
If affinity tags are added to polymeric tails, then specific binding to solid support is achieved, but the tagging process requires additional reagents and steps
Solution Approach 1:
The patent combines the tagging reagents (terminal transferase and modified dNTPs) with the solid support in a single reaction mixture. The tagging and binding occur in one pot without separate steps, reducing procedural complexity while maintaining high specificity through the affinity tag-mediated binding
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 ensures high specificity and efficiency in isolating nucleic acids from various biological samples, minimizing loss and allowing for repeated use of the bound nucleic acids in multiple assays, reducing the need for extensive purification and wash steps, and enabling the generation of cDNA libraries and biobanks.
Implementation Method 1
The reaction mix comprises terminal transferase (TdT), dTTP and dUTP, and wherein dUTP is biotinylated to form a polymeric tail incorporated with biotin
Implementation Method 2
The surface of the solid support is coated with avidin, streptavidin, or neutravidin
Data Source
AI summary
Disclosed is a novel technique of directly isolating nucleic acids from a biological sample and use of the isolated nucleic acid complexes for various applications and assays such as biobanking and sequencing.