eBook: Optimizing Results from Nucleic Acid Isolation - 5

challenges exist in the physical recovery of cfDNA
and in extracting as much relevant diagnostic data
as possible from fragmented, low-level nucleotides.

predicting disease recurrence, assessing resistance
to treatment (e.g., antibiotics, antitumor drugs), and
prenatal testing. Since fetal cells also release cfDNA,
associated methods are now available for cell-free
prenatal testing.

Kits and protocols

Liquid biopsies eliminate painful, costly surgical
biopsies and allow multiple or serial sample draws
without harming patients. Since these samples may
be taken from multiple tumor sites, liquid biopsies
can be utilized to map the rapidity and locations of
tumor spread.

Nucleic acid extraction methods have undergone
constant improvement since researchers first observed genetic mutations in cfDNA and realized its
potential significance as a diagnostic. From a sourcing perspective, plasma is preferred over serum for
tumor cfDNA analysis. Serum also contains cfDNA,
but lysis of healthy blood cells during serum sample
prep could interfere with the tumor DNA signal.

The foundational premise of liquid biopsies is that
ctDNA fragments incorporate the same mutations
as the tissue, whether cancerous or normal, from
which the ctDNA originated. This was demonstrated
in the mid-1990s for plasma-derived DNA associated
with cystic fibrosis and K-ras genes and was subsequently corroborated for many other conditions as
well-for example, loss of heterozygosity, promoter
hypermethylation, and single-nucleotide mutations.

Isolating cell-free nucleic acids from plasma does
not rely as much on cell lysis as tissue-based studies,
but it presents unique challenges nonetheless. Red
blood cells lack DNA, so they cannot contribute to
the background noise when analyzing blood fractions-but coagulation can interfere with both sample digestion and PCR amplification.

cfDNA concentrations correlate well with tumor
burden, but they are quite low compared with other
genes present-as little as 0.01% of the total cfDNA. Thus, despite the promise of liquid biopsies,

Numerous vendors offer kits for cfDNA isolation.
Most work quite well, but the multiplicity of methods and tools raises issues of reproducibility and

https://bmcresnotes.biomedcentral.com/articles/10.1186/s13104-018-3866-8 https://www.ncbi.nlm.nih.gov/pubmed/14578335 https://pdfs.semanticscholar.org/7065/dc8b9620328ccd1d80b3768b70d65fba93de.pdf https://pdfs.semanticscholar.org/7065/dc8b9620328ccd1d80b3768b70d65fba93de.pdf https://www.repository.cam.ac.uk/bitstream/handle/1810/262102/wan_et_al-2017-nature_reviews_cancer-aam.pdf;jsessionid=7270621BD97F50CF223CED9A0F734B53?sequence=4 https://www.ncbi.nlm.nih.gov/pubmed/8118388 https://www.nature.com/articles/nm0996-1035 https://www.ncbi.nlm.nih.gov/pubmed/10376981 https://www.ncbi.nlm.nih.gov/pubmed/10376981 https://www.ncbi.nlm.nih.gov/pubmed/22649089 https://www.nature.com/news/cancer-biomarkers-written-in-blood-1.15624 https://www.nature.com/news/cancer-biomarkers-written-in-blood-1.15624

eBook: Optimizing Results from Nucleic Acid Isolation

Table of Contents for the Digital Edition of eBook: Optimizing Results from Nucleic Acid Isolation