cfDNA (cell free DNA) refers to highly fragmented DNA that exists in human blood circulation and is free outside cells. In certain circumstances (such as cancer patients, pregnant women, patients receiving organ transplants, etc.), a small amount of cfDNA from "heterologous" cells (such as tumor cells, fetal cells, or donor cells) can be used as a marker for genetic testing things. The fragmented genomic DNA released after the rupture of dead tumor cells is also called ctDNA (circulating tumor DNA). "Liquid biopsy" technology based on cfDNA detection has received great attention in many aspects such as prenatal diagnosis, tumor screening, early diagnosis, treatment monitoring and prognosis assessment.

The "DNA ladder"-style fragmented nucleic acids produced during active cell lysis (also called apoptosis) are basically around 150-200bp in size. The human body is a large and complex circulatory system, in which the operations start over and over again. In addition to the large circulation of the entire human body, there are also "small cycles" of each component that makes up the human body. Simply put, it is the cycle of replacement states of various organs of the human body. Under normal circumstances, the human body will replace 98% of the cells in the body's tissues within half a year, which means that cells are constantly dying and new cells are being formed in our bodies every moment of every day.

Passive cell lysis - there are many ways of this lysis, which can come from tissue necrosis caused by external physical or chemical stimulation, or from the killing effect of the own immune system on cells. The difference is this The nucleic acids produced by the process are very large, approaching the size of genomic DNA.

Method 1: Phenol-Chloroform

Principle: Separate DNA from other impurities using the method that nucleic acid DNA is insoluble in organic solvents

Advantages: Low cost; equipment Low requirements.

Disadvantages: The operation is cumbersome; the recovery rate is low; there are many inhibitor residues; the reagents contain toxic components.

Method 2: Silica gel column

Principle: Nucleic acid is combined with the silica gel membrane under high salt conditions. The nucleic acid is eluted from the silica gel membrane under low salt and high pH values. Collect by centrifuging tubes multiple times

Advantages: High purity; low cost.

Disadvantages: Requires multiple high-speed centrifugations and many operating steps; negative pressure suction and open operation, low efficiency and prone to cross-contamination;

The adsorption column will be clogged; The recovery rate is low, the loss is large, and it is not suitable for small fragment extraction; the storage conditions of different reagents are inconsistent and easy to be lost.

Method 3: Magnetic Bead Method

Principle: Refined superparamagnetic nanosilica magnetic beads specifically adsorb free nucleic acids in the blood under saline solution conditions, and the magnetic beads that adsorb nucleic acids will Transfer the beads to low-salt conditions to elute them.

Advantages: High purity; high recovery rate, small fragments can also be purified; can be automated.

Disadvantages: The cost of imported reagents from abroad is high.

1. Detect the size of cfDNA fragments

a) Use gel electrophoresis to detect, but the fragment size cannot be accurately detected;

b) Use qPCR to detect, but only It can detect pre-selected sites and is not suitable for genome-wide analysis;

c) Use electron microscopy for detection, but it is time-consuming, laborious and low-efficiency;

d) Use large-scale sequencing Platform detection;

2. cfDNA-related molecular detection

Detection is carried out by calculating windowed protection scores (windowedprotectionscore, abbreviated as wps), that is: fragment size = a special fragment in the genome Number of DNA fragments spanning 120 bp within a point - the number of fragments with endpoints. It is related to nucleosome distance. We can infer the source of cfDNA based on the distance between nucleosomes.

3. Testing purpose

a) Detecting the size of cfDNA can tell which cell death mechanism it is produced by: ~10000bp cfDNA is produced by cell necrosis; 140-200bp cfDNA is produced by apoptosis; 313-798bp is pregnant women’s cfDNA;

b) Detecting cfDNA-related molecular signals, such as methylation, and nucleosome distance can trace its tissue origin;

c) Detecting changes in the number of cfDNA types of specific sequences can determine cancer and related diseases.

Blood cfDNA has important potential value in early diagnosis, prognosis, and monitoring of diseases. Its specific medical applications generally include the following aspects:

a) for prenatal diagnosis;

b) for condition analysis and efficacy observation of non-tumor diseases such as immune diseases;

c) For tumor-related analysis.

Among the above three types of applications, its value in tumor analysis is particularly important. Although cfDNA analysis in blood has not yet been listed as a clinically necessary detection indicator, thousands of research papers and a large number of The data from phase 2 and phase 2 clinical trials strongly supports the huge application value of this new technology in tumor prevention and treatment:

a) Early diagnosis of tumors;

b) Tumor treatment plan Determine;

c) Tumor efficacy observation;

d) Tumor prognosis assessment;

e) Tumor metastasis risk analysis;

f ) Tumor recurrence monitoring.

Wei Yi | Copywriter