Sequential Cloning System Using Portable Marker Segments
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Solution Overview
Problem
Conventional cloning systems for assembling chimeric constructs with multiple heterologous nucleic acid sequences are inefficient due to issues like inefficient restriction enzyme cleavage, ligation, and high background of non-recombinant host cells, requiring extensive screening and time-consuming purification steps.
Innovation Solution
A cloning system that uses a portable segment with a marker sequence to chaperone nucleic acid sequences into recipient constructs, allowing for sequential cloning by identifying recombinant cells based on the marker sequence, reducing the need for extensive screening and purification.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional cloning strategy is used with selectable marker gene in recipient vector, then recombinant vectors can be identified through resistance to selective agent, but extensive screening of host cells is required due to high background of non-recombinant host cells (99%)
Solution Approach 1:
The patent introduces a portable segment containing a marker sequence as an intermediary element that facilitates the identification of recombinant cells. This marker sequence acts as a mediator between the insert nucleic acid sequence and the host cell, providing a reliable indicator of successful cloning without requiring extensive screening of non-recombinant cells.
Solution Approach 2:
The cloning system divides the recipient vector into two functional parts: the essential backbone (origin of replication, selectable marker) and the portable segment (marker sequence for identification). This segmentation allows the marker sequence to be independently tracked, enabling precise identification of recombinant cells while reducing background noise from non-recombinant host cells.
2Reliability
If purification of fragment and/or linearized vector is performed before ligation to reduce background, then non-recombinant host cell background is reduced, but time-consuming steps are required and ligation efficiency is reduced by trace amounts of purification agents
Solution Approach 1:
The portable segment with its unique marker sequence serves as an intermediary that enables direct identification of successful ligation events. This eliminates the need for time-consuming purification steps to reduce background, as the marker sequence provides immediate identification of recombinant cells among the transformation mixture.
Solution Approach 2:
The cloning system uses the marker sequence within the portable segment to self-identify successful recombinant events. This self-service mechanism eliminates the need for external purification steps or extensive screening, allowing direct transformation and identification of recombinant cells based on the marker sequence.
3Stability of the object's composition
If donor vector with its own origin of replication and selectable marker gene is used, then insert nucleic acid sequence can be maintained, but ligation to donor vector backbone occurs reducing efficiency of sequential cloning
Solution Approach 1:
The patent extracts the marker sequence function from the complete donor vector system, creating a portable segment that contains only the essential marker sequence without the donor vector's origin of replication or selectable marker gene. This extraction prevents unwanted ligation to donor vector backbone while maintaining the insert sequence's stability through the portable segment's marker sequence.
Solution Approach 2:
The system segments the donor vector components, separating the marker sequence (portable segment) from the origin of replication and selectable marker gene (recipient vector components). This segmentation prevents ligation between donor and recipient vectors while maintaining insert stability through the portable marker segment.
Data Source
AI summary
This invention discloses a cloning system and more particularly a system for sequentially cloning a plurality of heterologous nucleic acid sequences to assemble a chimeric construct of interest. The cloning system employs a marker sequence, which confers an identifiable characteristic on host cells in which it is contained, to chaperone individual insert nucleic acid sequences into recipient constructs that do not comprise the marker sequence but comprise other nucleic acid sequences for inclusion in the chimeric construct. Recombinant constructs into which one or more insert nucleic acid sequences have been introduced with the chaperone marker sequence are isolated by introducing recombinant constructs into host cells and identifying hosts cells with the identifiable characteristic.