Capillary Blood cfDNA Isolation via Silica Membrane
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Solution Overview
Problem
Current liquid biopsy techniques require large volumes of venous peripheral blood and complex processing, limiting their clinical application due to high operational time and cost, and there is a need for a more efficient method to detect circulating tumor DNA for cancer screening.
Innovation Solution
A method for isolating circulating free nucleic acids from capillary blood using a silica membrane column, involving dilution, lysis, binding, washing, and elution, followed by constructing a sequencing library for next-generation sequencing, which allows for low-volume, low-cost cancer screening.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If conventional liquid biopsy techniques are used with venous peripheral blood, then sufficient cfDNA can be obtained for detection, but the sample volume required is large (at least 10 mL) and the processing is complex
Solution Approach 1:
The patent changes the sample type from venous blood to capillary blood, and modifies processing parameters including dilution ratio (1:2 with PBS), lysis buffer composition (ACL buffer with protease K), and centrifugation conditions (16,000 ×g for 10 minutes). These parameter changes enable sufficient cfDNA extraction from small 500 μL capillary blood samples, resolving the contradiction between obtaining adequate cfDNA and simplifying the operation.
2Quantity of substance
If large volume venous blood is processed by centrifugation, then plasma can be separated, but large centrifuges with limited channels (6-8) are required and operational time is long
Solution Approach 1:
The patent segments the processing into two centrifugation steps: first centrifugation at 1,600 ×g for 10 minutes to separate plasma from cells, then second centrifugation at 16,000 ×g for 10 minutes to remove cell debris. This segmentation allows use of smaller centrifuges with more channels (up to 48 channels mentioned), significantly improving throughput and reducing operational time while still obtaining sufficient plasma volume.
3Ease of operation
If capillary blood sample is used, then blood collection is simple and sample volume is reduced, but the amount of cfDNA obtained may be insufficient for detection
Solution Approach 1:
The patent applies preliminary action by diluting the capillary blood sample 1:2 with PBS before processing. This pre-dilution step increases the effective volume for cfDNA extraction while removing excess salts and proteins that could interfere with downstream processing. Combined with optimized lysis conditions (ACL buffer, protease K, 60°C for 30 minutes) and double centrifugation, this preliminary action ensures sufficient cfDNA is obtained from the small 500 μL capillary blood sample.
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 enables efficient isolation and sequencing of circulating free nucleic acids from small capillary blood samples, reducing operational time and cost, while maintaining the sensitivity and accuracy of cancer detection, facilitating real-time monitoring of cancer dynamics.
Implementation Method 1
loading the mixture in step (3) to a silica membrane column, to allow the binding of DNA to the silica membrane column
Data Source
AI summary
The disclosure relates to a method for separating circulating free nucleic acid (e.g., circulating free DNA, cfDNA) from capillary blood sample (e.g., capillary blood plasma) and methods of determining and/or assessing the risk of cancer in a subject.