190
- Refrigerated centrifuge capable of at least 14,000 × g.
- Refrigerated ultracentrifuge capable of at least 105,000 × g.
- 0.22 μm filter and syringe to drive filtration.
- Phospho-buffered saline.
- Chromatographic separation column (e.g., Tricorn 5/20
Column, GE Lifesciences). - Sepharose 2B beads.
- Spectrophotometer.
- Agitating waterbath.
- [Optional] Liquid nitrogen.
- Extraction Buffer: 10 mM Tris–HCl (pH 8), 10 mM ethylene-
diaminetetraacetic acid (EDTA), 30 mM NaCl, 1 mM CaCl2,
0.5% sodium dodecylsulfate. - Extraction Buffer for exosomes: 10 mM Tris–HCl (pH 8),
10 mM EDTA, 30 mM NaCl, 1 mM CaCl2, 1% sodium
dodecylsulfate.
3 Methods
See Fig. 1 for a schematic representation of the basic method.
Isolation of ctDNA from plasma samples is a relatively simple
procedure. The main considerations are to minimize lysis of intact
benign cells and to avoid contaminating the plasma with any cells
from the blood. While neither of these events will prevent the
detection of ctDNA, the presence of contaminating DNA from
lysed or contaminating cells will potentially make detection more
difficult as normal DNA from these sources may swamp signal
from ctDNA. This is especially important in applications where
detection/quantitation of heterogeneous mutations is needed.
Blood should be treated gently, and initial centrifugation and
plasma isolation should take place as soon as possible after blood
collection.
The other main concern for this method is that protein from
blood plasma be sufficiently degraded to be removed in the etha-
nol precipitation step and that DNA bound proteins, such as his-
tones, are destroyed and release DNA to maximize yields.
Incubation time for proteinase K may need to be adjusted to suit
the needs and sensitivities of downstream procedures.- Collect blood in a vacutainer or similar vessel impregnated
with EDTA, lithium heparin, or other anticoagulant. - Sufficient blood should be collected to maximize plasma yield,
and hence overall DNA produced (see Note 1).
3.1 Isolation
of Circulating Tumor
DNA from Plasma
3.1.1 Plasma Separation
Robert A. Smith and Alfred K. Lam