qPCR Amplicon-Length Assay for Plasma Separation Efficiency
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Existing methods for evaluating plasma separation from whole blood are laborious, expensive, and insufficiently accurate, and require large DNA amounts for gel electrophoresis, which is a challenge when working with circulating cell-free DNA.
Innovation Solution
A real-time PCR assay using co-amplification of a short amplicon (70-150 bps) and a long amplicon (350-600 bps) to determine separation efficiency based on the difference in amplification levels, eliminating the need for absolute quantification and standard curves.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If gel electrophoresis is used to determine separation efficiency by detecting long versus short DNA fragments, then separation efficiency can be evaluated, but large amounts of DNA are required which is a major difficulty when working with circulating cell-free DNA
Solution Approach 1:
The invention changes the parameter of amplicon length to differentiate between cfDNA and gDNA. By designing qPCR assays with specific amplicon sizes (short amplicons for cfDNA, long amplicons for gDNA), the method enables sensitive detection of separation efficiency without requiring large DNA amounts, thus resolving the contradiction between measurement precision and quantity requirement
Solution Approach 2:
The invention replaces the mechanical gel electrophoresis system with a biochemical qPCR system. Instead of physically separating DNA fragments by size through electrophoresis and visualizing them, the method uses differential amplification of short and long amplicons followed by fluorescent detection, achieving the same separation efficiency evaluation with much lower DNA requirements
2Measurement precision
If blood cell counts are performed manually using a hemocytometer or automatically using a flow cytometer to evaluate separation efficiency, then separation efficiency can be assessed, but the methods are laborious and expensive
Solution Approach 1:
The invention extracts only the necessary information for separation efficiency assessment by directly measuring DNA fragment sizes in the plasma sample through qPCR, rather than performing complete blood cell counts. This approach takes out the essential measurement (DNA contamination level) from the complex blood cell counting process, making the procedure simpler and less expensive while maintaining assessment accuracy
Solution Approach 2:
The invention creates a simplified copy of the separation efficiency assessment by using qPCR amplification to represent the presence of long DNA fragments (gDNA contamination) without needing to actually count or identify individual blood cells. The amplification products serve as copies that indicate the level of contamination, enabling easy and inexpensive evaluation
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
Provides a simple, cost-effective, and accurate method to assess plasma separation efficiency without laborious steps, sensitive to noise factors, and applicable to any DNA concentration.
Implementation Method 1
A real-time PCR assay using co-amplification of a short amplicon (70-150 bps) and a long amplicon (350-600 bps) to determine separation efficiency based on the difference in amplification levels
Data Source
AI summary
The present invention provides according to some aspects methods and kits for determining the efficiency of plasma separation from whole blood using quantitative PCR amplification of two amplicons, namely, a short amplicon of e.g. 70-150 bps and a long amplicon of e.g. 350-600 bps. The separation efficiency is determined based on the difference in amplification levels of the two amplicons. Advantageously, the separation efficiency is determined without absolute quantification of DNA and/or determination of copy number of any gene/locus.