Multiple-control DNA Calibrator Plasmids for Multiplex Quantitation
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Solution Overview
Problem
Generating precise calibration data for multiplex DNA quantitation is challenging due to inaccuracies in measuring nucleic acid concentrations and differences in nucleic acid configurations, such as supercoiled plasmids, genomic DNA, or fragmented DNA, which affect amplification efficiency.
Innovation Solution
Development of calibrator DNA plasmids containing multiple control fragments separated by restriction endonuclease recognition sites, allowing for equimolar mixtures of target nucleic acids, and methods for their use in producing calibration standards for DNA quantitation, including bisulfite-converted DNA fragments and specific enzyme reactions.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If different target nucleic acid preparations are used to generate calibration curves for multiplex detection, then calibration data can be obtained for multiple targets, but inaccuracies in concentration measurement and difficulty in producing precise equimolar mixtures occur
Solution Approach 1:
The calibrator nucleic acid is divided into multiple separate control fragments, each corresponding to a different amplification target. These fragments are serially linked in a specific order within a single nucleic acid molecule, allowing them to be processed independently during amplification while maintaining known relative quantities.
Solution Approach 2:
A single calibrator nucleic acid molecule serves multiple functions by containing multiple control fragments that correspond to different amplification targets. This universal calibrator can be used for multiplex detection of multiple targets simultaneously, eliminating the need for separate calibrator preparations for each target.
2Adaptability or versatility
If different target nucleic acid configurations (supercoiled plasmid, genomic DNA, fragmented DNA) are used, then various target types can be calibrated, but amplification efficiency varies due to configuration differences
Solution Approach 1:
Each control fragment within the calibrator nucleic acid is designed with specific local characteristics that match the corresponding target nucleic acid sequence. The fragments are arranged in a defined linear order and can be configured to match the specific properties (length, sequence composition, secondary structure) of the targets they represent, ensuring consistent amplification efficiency.
3Measurement precision
If serial dilution of calibrator nucleic acid is performed to generate calibration curves, then quantitative analysis can be achieved, but time-consuming multiple preparation steps are required
Solution Approach 1:
The control fragments are pre-arranged in a specific serial order within the calibrator nucleic acid molecule during construction. This preliminary arrangement of fragments in defined positions eliminates the need for time-consuming serial dilution steps during calibration curve generation, as the relative quantities are already established by the physical arrangement of fragments.
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
Enables accurate and reliable DNA quantitation by providing stable calibration standards that account for variations in nucleic acid configurations, improving the precision of multiplex DNA quantitation reactions.
Implementation Method 1
the control portion comprises two or more serially-linked control fragments separated by restriction endonuclease recognition sites
Data Source
AI summary
The present invention provides DNA plasmids that find use as calibrators or reference standards for calculating DNA quantities in a sample. In particular, provided herein are DNA plasmids that contain multiple control fragments, and methods of their use in DNA quantitation.