eBook: Optimizing Results from Nucleic Acid Isolation - 4

Challenges in cfDNA
Sample Preparation
Find out how to extract as much relevant diagnostic data
as possible from fragmented, low-level nucleotides.
Angelo DePalma, Ph.D.
acids, allowing contaminants to wash through while
retaining genetic material.

DNA isolation from cells, tissues, and bodily fluids is
the gateway operation to innumerable downstream
applications in drug discovery, medicine, and basic
research. Given the high quality of genetic materials from whole blood and plasma, amplification
through polymerase chain reaction (PCR) or multiple displacement amplification (MDA) provides a
nearly limitless supply of DNA for routine medical
diagnostics, prenatal testing, and companion diagnostics, as well as for monitoring drug efficacy and
disease progression.

Cell-free DNA and liquid biopsies
For cell-free preparations from blood or plasma,
cellular components are first pelleted, and the nucleic acids are extracted directly from the supernatant. Quantification occurs through a combination
of an enrichment or depletion step to raise target
gene concentrations, qPCR amplification, and analysis by capillary electrophoresis or next-generation
sequencing.

DNA (or RNA) extraction involves chemical, mechanical, or enzymatic cell disruption and protein
denaturation, the choice of which depends on the
sample and the downstream applications. Protocols
incorporating enzymes and detergents are considered "gentle," whereas mechanical homogenization
is more vigorous.

Cell-free DNA (cfDNA) preps have assumed a special significance with the emergence of molecular
diagnostics.
Both healthy and diseased cells shed DNA when
they die or are stressed. This genetic material, which
includes circulating tumor DNA, consists of short nucleic acid fragments found in all bodily fluids and is
released by both normal and diseased cells.

Disruption is typically followed by either phenol-chloroform or solid-phase oligonucleotide extraction, both of which isolate DNA while removing
molecular and cell-derived contaminants. Columnbased methods employ chaotropic salts that break
hydrogen bonds between contaminants and nucleic

Diagnostics based on tumor-derived cfDNA, known
as "liquid biopsies," have great potential for early cancer detection, genetic and epigenetic monitoring,

https://bmcresnotes.biomedcentral.com/articles/10.1186/s13104-018-3866-8

eBook: Optimizing Results from Nucleic Acid Isolation

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