Frameshift Mutation Detection Using Complementary Counter-Strand Reporter Fusion
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Solution Overview
Problem
Existing methods for determining frameshift mutations in coding nucleic acids are limited, as they require the gene to be examined to have no internal stop codons, which is not feasible for many synthesized genes, and are prone to errors such as insertions and deletions that disrupt the reading frame.
Innovation Solution
A method using a host cell with a double-stranded nucleic acid comprising a target nucleic acid and a complementary counter-strand nucleic acid optimized to lack stop codons, where the counter-strand is operatively linked to a reporter gene, allowing expression to indicate the absence of frameshift mutations, even in the presence of internal stop codons.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If the existing method using a reporter gene is used to determine frameshift mutations, then the reading frame can be verified, but the gene cannot contain internal stop codons which limits applicability
Solution Approach 1:
The patent inverts the conventional approach by using the complementary strand (antisense strand) instead of the target gene sequence itself. The complementary strand is ligated to the reporter gene in reverse orientation, so that its sequence runs 5' to 3' in the correct reading frame direction. This inversion allows genes with internal stop codons to be analyzed because the complementary strand's stop codons correspond to different positions and do not interfere with the reporter gene expression assay.
2Measurement precision
If the target nucleic acid is directly used with reporter gene, then frameshift can be detected, but internal stop codons disrupt the reading frame and prevent detection
Solution Approach 1:
The patent creates a copy of the target nucleic acid in the form of its complementary strand. This complementary strand serves as a surrogate that preserves the mutation information (frameshifts) but presents the sequence in a format suitable for reporter gene fusion. The complementary strand is synthesized or generated and then ligated to the reporter gene, allowing indirect detection of frameshift mutations in the original target sequence without the interference of internal stop codons.
3Quantity of substance
If gene length increases, then more information is available, but probability of mutations increases sharply
Solution Approach 1:
The patent implements a feedback mechanism where the expression level of the reporter gene provides information about the integrity of the complementary strand sequence. If frameshift mutations are present in the target gene, they will be reflected in the complementary strand and prevent proper reporter gene expression. This feedback allows for the identification and selection of correctly synthesized genes, enabling quality control in gene synthesis processes even for long genes where mutation probability is high.
Data Source
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AI summary
The invention relates to a method for determining frameshift mutations in coding nucleic acid sequences.