Corrected Sizing Ladder for Automated DNA Fragment Isolation
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current methods for separating and isolating DNA and protein fragments from electrophoretic separation matrices are time-consuming, laborious, and potentially damaging to the samples, and require manual excision and extraction, which can lead to DNA nicking under UV light.
Innovation Solution
A method using a corrected sizing ladder for real-time sizing analysis in fractionation devices, where archival or real-time migration times are adjusted using correction factors to improve the accuracy and precision of isolating sample components, allowing for automated diversion of desired fragments to a collection location based on size determination.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If manual excision and extraction methods are used to isolate DNA fragments from separation matrices, then fragment isolation can be achieved, but the process becomes time-consuming and laborious
Solution Approach 1:
The patent replaces manual mechanical excision methods with automated detection and collection systems. The system uses a detector to identify fragment positions and an automated mechanism to collect fragments at specific locations, eliminating the need for manual razor blade excision and chemical extraction processes.
Solution Approach 2:
The separation matrix is designed to allow fragments to be collected directly from the gel structure without requiring its complete removal or dissolution. The system utilizes the gel's own structure as the separation medium while enabling automated fragment collection, making the process self-sufficient without additional extraction steps.
2Difficulty of detecting and measuring
If UV light is used to illuminate fragments for visualization during manual excision, then fragment locations can be identified, but DNA nicking and damage can occur
Solution Approach 1:
The patent replaces UV light illumination with alternative detection methods such as fluorescent labeling or other non-damaging visualization techniques. The detector system identifies fragment positions without requiring prolonged UV exposure, thereby eliminating DNA nicking while maintaining the ability to locate and collect fragments.
3Measurement precision
If standard sizing ladders are used for fragment size determination, then sizing analysis can be performed, but batch-to-batch variations in sieving gel affect measurement precision
Solution Approach 1:
The patent incorporates a feedback mechanism where the system detects the actual migration positions of sizing ladder fragments and uses this information to calibrate and correct the size determination for sample fragments. This real-time calibration compensates for batch-to-batch gel variations and ensures accurate size measurement regardless of gel consistency differences.
Solution Approach 2:
The system adjusts sizing parameters based on actual experimental conditions by comparing observed ladder fragment positions with expected positions. This dynamic parameter adjustment compensates for variations in sieving gel properties across different batches, maintaining measurement precision despite changes in gel composition or manufacturing variations.
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 enhances the efficiency and accuracy of sample component isolation by enabling real-time sizing and automated diversion, reducing the risk of sample damage and labor, and improving the precision of fractionation in electrophoretic separations.
Implementation Method 1
a voltage gradient is applied along the length of the channel. As the sample components (also referred to as 'fragments') are electrophoretically transported along the length of the channel and through the separation (sieving) matrix disposed therein
Implementation Method 2
those components are resolved. The separated components are then detected at a detection point along the length of the channel
Implementation Method 3
the separated fragments are stained and illuminated by shining ultraviolet (UV) light on the fragments to visualize the separated bands
Data Source
Figure 1
Figure 2
Figure 3
AI summary
The invention provides methods of performing a sizing analysis. The methods comprise the correction of a sizing ladder which contains components of a known size and relates component size to migration time, and which is used in performing the sizing analysis. In one method, the sizing ladder is corrected for batch-to-batch variations in a sieving gel. In another method, the sizing ladder is corrected using a standard marker in a sample. The methods may be used individually or in combination.