Isolation of cell free / circulating tumor DNA from plasma

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Identification of biomarkers that indicate presence of disease are highly sought after. Non-invasive methods to measure those biomarkers are even more valuable. By extracting and measuring cell-free DNA, scientists have satisfy both.

Cell free DNA are degraded fragments released in plasma. Elevated levels of cfDNA are found in cancer states, making assessment of somatic genomic alterations from tumors possible using sequencing. Cell free fetal DNA (cffDNA) can be found as early as 7 weeks gestation, and analysis of cffDNA is already being used in non-invasive prenatal diagnostics. Cell free DNA (cfDNA) in blood was first described by Mandel and Metais in 1948 [1] but only recently has been identified as having utility for prenatal testing and disease diagnostics and monitoring.

Unlike mutations that are passed from a parent to child and are in every cell of your body, somatic mutations form during a person’s life. These somatic mutations are present in tumor cell DNA and are an excellent biomarker if they can be measured and monitored.

Acquiring tumor DNA often requires a biopsy, a potentially risky and invasive procedure. In many cases presence of a tumor or the ability to biopsy is not even an option for patient. During tumor turnover and progression, apoptotic and necrotic cells release small pieces of their DNA (cfDNA) into the bloodstream. The amount of cfDNA in the blood steam is influenced by clearance and filtering of the blood and lymphatic circulation.

Detecting cfDNA in plasma is called a ‘liquid biopsy’ and is already a popular method for obtaining clinical samples for prenatal testing, disease diagnostics and monitoring. One of the challenges of liquid biopsies, are standardization of the isolation procedure and maintaining  uniform specificity and sensitivity. Extraction of cfDNA can be carried out using magnetic beads or silica matrices along with chaotrophic salts, such as guanidine thiocyanate. While several commercial approaches (Table 1) exist, none have undergone rigorous large patient scale studies. Once more information is known, universal standardization should allow greater clinical utility.

Commercial kits for extraction of cfDNA need to be designed to extract uniform DNA copies from varying biopsy volumes. Scalability and adaptability for cell free fetal and ctDNA are important considerations. Below we highlight current kits available in the market. In a future blog post we’ll discuss isolation and sequencing standardizations required for broader use of cfDNA liquid biopsy.

Table 1.

Kit Company Method Digestion Prep Time (min) Plasma Volume (mL) Elution (uL) DNA sizes

(bp)

NextPrep-Mag

 

Bioo Scientific Mag Beads Proteinase K (optional) 30 1 – >5 12 >50
Chemagic cfNA Chemagen Mag Beads Proteinase K 120 2 – 10 60 >100
MagMAX Cell Free DNA Kit Thermo Fisher Mag Beads Proteinase K

(optional)

40 1 – >5 15 >50
QIAamp

 

Qiagen Column Proteinase K 120 1-5 20 >70
Quick-cfDNA

 

Zymo Research Column Proteinase K 60 3- 10 35 >100

One thought on “Isolation of cell free / circulating tumor DNA from plasma

  1. The Thermo Fisher kit is called MagMAX Cell-free DNA Kit. It can scale up to any volume. We did 250 mL extraction in a bottle. The cut off is >= 50bp.

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