Anchor Oligonucleotide Tag Insertion in Nucleic Acid Synthesis
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Solution Overview
Problem
Current methods for inserting tag sequences into nucleic acids are complex and require multiple steps, including synthesis of the opposite strand, which is time-consuming and laborious.
Innovation Solution
A method involving the provision of a template nucleic acid, hybridization of an anchor sequence with the template, and synthesis of a new strand that complements the anchor oligonucleotide, allowing for direct insertion of a tag sequence at the 3' end during the first polymerase reaction without subsequent synthesis of the opposite strand.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If traditional methods (template-independent polymerase, template-switching primer, or external primer attachment) are used to insert tag sequences, then tag sequence insertion is achieved, but the process requires multiple enzymatic reactions and synthesis of the opposite strand, making it time-consuming and laborious
Solution Approach 1:
The patent merges the tag sequence insertion step with the first-strand synthesis reaction by incorporating the template tag sequence directly into the anchor oligonucleotide. This eliminates the need for separate enzymatic reactions (template-independent polymerase, template-switching, or external primer attachment) and directly integrates tag insertion into the initial polymerase reaction, thereby reducing time and labor while maintaining accuracy
Solution Approach 2:
The template tag sequence is preliminarily incorporated into the anchor oligonucleotide before the synthesis reaction begins. This preliminary design allows the polymerase to directly copy the template tag sequence during first-strand synthesis, eliminating the need for subsequent actions to add tag sequences, thus saving time and reducing procedural complexity
2Manufacturing precision
If template-independent polymerase is used to attach homopolymer, then tag sequence can be inserted, but an additional template-independent polymerase reaction is required, increasing process complexity
Solution Approach 1:
The patent combines the tag sequence insertion function with the standard polymerase reaction by using a template tag sequence in the anchor oligonucleotide. This eliminates the need for a separate template-independent polymerase reaction, reducing the number of enzymatic steps from two (standard polymerase + template-independent polymerase) to one (standard polymerase only), thereby simplifying the overall process
Solution Approach 2:
The anchor oligonucleotide with template tag sequence serves multiple functions: it provides the anchor for first-strand synthesis initiation and simultaneously serves as the template for tag sequence insertion. This multi-functionality eliminates the need for specialized template-independent polymerase, allowing a single polymerase to perform both synthesis and tag insertion
3Manufacturing precision
If template-switching primer is used, then tag sequence is inserted at mRNA cap end, but the method depends on specific reverse transcriptase properties and requires cytosine nucleotide attachment, increasing procedural steps
Solution Approach 1:
The patent extracts the tag sequence insertion step from the complex template-switching mechanism. Instead of relying on reverse transcriptase's specific property to attach cytosine nucleotides and then hybridize with G-containing oligonucleotides, the invention directly incorporates the template tag sequence into the anchor oligonucleotide, allowing straightforward copying by polymerase without specialized enzyme properties or additional hybridization steps
Solution Approach 2:
Instead of using template-switching where the polymerase switches templates during synthesis, the invention inverts the approach by pre-incorporating the template tag sequence into the anchor oligonucleotide that remains throughout the reaction. This inversion eliminates the need for template switching while achieving the same tag insertion result with simpler procedure
4Manufacturing precision
If external primer with tag sequence is used, then tag sequence is inserted at 5' end, but synthesis of opposite strand is still necessary to insert complementary sequence at 3' end, maintaining multi-step process
Solution Approach 1:
The patent merges the tag sequence insertion at the 3' end with the first-strand synthesis reaction itself. By incorporating the template tag sequence into the anchor oligonucleotide, the tag sequence is directly inserted during the initial polymerase reaction without requiring separate opposite strand synthesis, thereby improving productivity while maintaining accuracy
Solution Approach 2:
The template tag sequence is preliminarily designed into the anchor oligonucleotide before synthesis begins. This preliminary configuration allows the tag sequence to be inserted in a single step during first-strand synthesis, eliminating the need for subsequent opposite strand synthesis to add the complementary sequence, thus enhancing overall efficiency
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 simplifies the process of inserting tag sequences, saving time and resources by integrating the tag sequence into the first polymerase reaction, and allows for efficient insertion at the 3' end of nucleic acids during RNA or DNA synthesis.
Implementation Method 1
hybridization of at least one anchor sequence of at least one anchor oligonucleotide with at least one sequence section of the template nucleic acid
Implementation Method 2
synthesis of a new strand of nucleic acid, which partially complements the template nucleic acid and which contains a sequence complimentary to the non-hybridized portion of the anchor oligonucleotide
Data Source
AI summary
The present invention concerns a method for inserting one or more tag sequences into a nucleic acid characterized by the following steps: (a) preparation of a template nucleic acid; (b) hybridization of at least one anchor sequence of at least one anchor oligonucleotide with one sequence section of the template nucleic acid; and (c) synthesization of a new strand of nucleic acid, which is partially complementary to the template nucleic acid and which contains a sequence complementary to the non-hybridized portion of the anchor oligonucleotide, e.g. to at least one tag sequence, on its 3′ end.