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Circulating Tumor DNA: Most sensitive method for analyzing ctDNA
Circulating Tumor DNA: Most sensitive method for analyzing ctDNA. What is the most sensitive method for analyzing ctDNA?
ctDNA technology FAQ
Q1: Is plasma or serum more suitable for ctDNA separation?
A: A lot of research data shows that the content of cfDNA in serum is significantly higher (3-24 times) than that in plasma. This is mainly because the blood coagulation process may cause a small amount of hemolysis during serum preparation, so there will be a certain amount of blood cell nucleic acid contamination. However, there is currently no sufficient evidence to confirm whether there is such an imbalance in the content of ctDNA in serum and plasma.
Q2: Is there any special attention to the preparation of plasma/serum for ctDNA extraction?
A: The plasma/serum preparation process required for ctDNA extraction is different from conventional serum/plasma preparation and needs special attention:
Serum/plasma usually needs to be prepared by a “two-step centrifugation method”. The first centrifugation uses conventional serum/plasma preparation centrifugal speed, and the second step requires high speed (14000-16000g) to remove possible residues in plasma/serum. Cell debris and some genomic DNA released by broken cells.
The plasma/serum preparation process avoids hemolysis as much as possible, such as reducing the time from blood draw to plasma/serum preparation, and strictly controlling the centrifugal speed. Because the genomic DNA released by broken blood cells may greatly dilute the small amount of cfDNA, which affects the accuracy of subsequent detection.
Q3: How to control and quantify the extracted ctDNA?
A: The fragment distribution of ctDNA can be determined by Agilent2000 (2100).
In the case of extremely low ctDNA, its quantitative determination is extremely susceptible to contamination by a small amount of single-stranded nucleic acid, protein and RNA, so it may not be suitable for quantitative strategies that use ultraviolet absorption methods, such as NanoDrop. Relatively speaking, Qubit is mostly used for the quantification of cfDNA (Qubit®dsDNA HS assay).
Q4: What are the methods for analyzing ctDNA mutations?
A: The method of analyzing the mutation characteristics of ctDNA is different according to the type of mutation we are concerned about:
To be detected are known point mutations, the more common methods include BEAMing, Digitaldroplet PCR, etc.;
To be detected is a single or multiple gene mutations, the more common methods are SafeSeqs, TAM-Seq;
To be detected is genome chromosome copy number variation (CNV), with Digitalkaryotyping;
What is to be detected is the genome chromosome rearrangement, with PARE.
Q5: What is the most sensitive method for analyzing ctDNA?
A: The sensitivity of ctDNA analysis also depends on the features of ctDNA mutations of interest. For features such as chromatin recombination, studies have shown that the sensitivity can reach 0.002% (mutant type: wild type approximately 1:50,000) (Sci TranslMed. 2010 Feb 24;2(20):20ra14. doi: 10.1126/scitranslmed.3000702.) And a series of methods for point mutation detection, the most common are BEAMing, digitaldroplet PCR, etc., the sensitivity can reach 0.005-0.01%. The NGS sequencing technology that targets multiple genes has a detection sensitivity of about 0.1-0.01%.
Q6: Is there currently any special method to enrich ctDNA in cfDNA?
A: At present, due to the nature of ctDNA, apart from the fact that it carries mutations, we have limited understanding of other physical and chemical properties. However, a large number of studies suggest that ctDNA is mostly concentrated in fragments of 150-200bp. Therefore, it is theoretically possible to fractionate nucleic acid fragments of this size (Sizefractionation) to achieve the enrichment of ctDNA. There is already a commercialized “TheEpiQuik™ Circulating Cell-Free DNA Isolation Kit” for related kits, but cfDNA is still extracted.
(source:internet, reference only)