Nucleic Acid Cloning Vector Merging Heavy and Light Chains
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Solution Overview
Problem
Current methods for cloning nucleic acids, particularly for therapeutic proteins like antibodies, are laborious and not amenable to high-throughput or automated techniques due to complexities in reformatting and the need for multiple cloning steps, leading to inefficiencies in identifying correct sequences and expressing complete antibodies.
Innovation Solution
A method that allows for the cloning of nucleic acids encoding polypeptides into a single vector, using a marker protein for detection and isolation, enabling high-throughput and automated cloning by linking unlinked nucleic acids in a single cloning cycle without requiring multiple vectors or cloning cycles, and facilitating operable linking with promoters and enhancers for expression.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If multiple cloning steps and vectors are used to clone nucleic acids encoding antibody chains, then the correct sequences can be identified, but the process becomes laborious and not amenable to high-throughput or automated techniques
Solution Approach 1:
The patent combines multiple cloning steps into a single cloning cycle by using a single vector that can accommodate both heavy chain and light chain nucleic acids. The vector contains multiple cloning sites that allow simultaneous insertion of both chain encoding sequences, eliminating the need for sequential cloning operations and enabling high-throughput processing.
Solution Approach 2:
The expression vector is designed with universal features including multiple cloning sites that can accept various nucleic acid sequences encoding different antibody chains. The vector serves multiple functions: it can clone both heavy and light chains, provides selection markers, and enables expression in mammalian cells, thereby streamlining the entire cloning workflow.
2Adaptability or versatility
If multiple vectors are used to express antibody light chain and heavy chain separately, then each chain can be expressed, but the complexity increases making it difficult to perform reformatting in high throughput or automated manner
Solution Approach 1:
The patent merges the functionality of two separate vectors (light chain expression vector and heavy chain expression vector) into a single dual-purpose vector. This single vector contains all necessary elements including promoters, selection markers, and multiple cloning sites to accommodate both light chain and heavy chain nucleic acids, thereby reducing operational complexity.
Solution Approach 2:
The vector is segmented with distinct cloning sites and expression elements for light chain and heavy chain, allowing independent manipulation and insertion of each chain's nucleic acid sequence while maintaining them within a single vector framework. This segmentation enables flexible reformatting without requiring separate vector operations.
3Ease of manufacture
If restriction endonuclease-based technology is used for reformatting antibodies, then nucleic acids can be cloned, but the multiple cloning site in expression vector encodes additional amino acids that may have undesirable functional effects and form immunogenic epitopes
Solution Approach 1:
The patent employs local quality by placing restriction endonuclease sites and cloning sites in specific locations within the vector that do not interfere with the coding sequences of the antibody chains. The multiple cloning sites are positioned in non-coding regions or at termini where inserted nucleic acids can be seamlessly integrated without adding unwanted amino acids to the protein structure.
Solution Approach 2:
The patent extracts the cloning functionality from the coding regions by using separate multiple cloning sites that are spatially separated from the antibody chain encoding sequences. This allows nucleic acid insertion without disrupting the reading frames or adding unwanted amino acids to the functional regions of the antibody proteins.
4Measurement precision
If traditional cloning methods with solid medium are used to select for individual clones, then correct clones can be identified, but the process is time-consuming and not readily amenable to automation
Solution Approach 1:
The patent replaces the mechanical process of manual colony screening on solid media with automated liquid-phase processing and electronic detection systems. The vector includes selection markers that can be detected by automated instruments, enabling high-throughput identification of correct clones without manual intervention and significantly reducing the time required for screening.
Solution Approach 2:
The vector incorporates self-selecting features including antibiotic resistance markers and fluorescent tags that automatically identify correct clones without requiring manual screening. The selection markers provide self-service by enabling automated detection and isolation of the correct nucleic acid insertions through standard laboratory automation techniques.
Data Source
AI summary
The present disclosure provides methods for producing expression constructs comprising linking a plurality of unlinked nucleic acids, including a nucleic acid encoding a marker protein.