Cell-free DNA

First identified in the mid-twentieth century, cell-free DNA (cfDNA) promises to revolutionize clinical diagnostics.

AATI cfDNA Photo

What is cfDNA?

A key biomacromolecule in the growing area of liquid biopsies, cfDNA could provide non-invasive and safer screening procedures to assess fetal health, detect the early stages of various cancers and rejection of transplants. Found in various bodily fluids including serum, plasma, saliva, cerebral spinal fluid (CSF), and urine, cfDNA can be isolated from individuals in both healthy and various disease states. In healthy individuals, cfDNA is actively secreted from cells as DNA is continually synthesized an by cell turn over through apoptosis. cfDNA associated with disease states is released due to different types of cell death such as apoptosis, necrosis, autophagy, or mitotic catastrophe. Apoptosis activates endogenous endonucleases that cleave DNA in repeatable lengths. DNA is wrapped around nucleosomes like "beads on a string". The pattern of cfDNA cleavage is guided by the nucleosome positioning. Variations of 165 bp cfDNA lengths and multiples thereof are a result of the length of DNA wrapped around the nucleosome, the different DNA linker lengths between the nucleosomes, and degradation of the linkers between the nucleosomes.

Table 1: Sources of cfDNA from various disease states.
Blood Serum/Plasma Saliva CSF Fetus Placenta via Mother's Serum Urine
Tumor X X X
Autoimmune Disorders
Lupus X
Rheumatoid Arthritis X
Newborn Genetic Mutation
Down Syndrome (trisomy 21) Mutation X
Edwards Syndrome (trisomy 18) Mutation X
Patau Syndrome (trisomy 13) Mutation X
Parasitic Infection X X
Inflammation (Aging) X
Exercise X X
Transplant/Graft Rejection X X
Trauma
Brain Injury X X
Skin Burns X

Separation of cfDNA often displays three fragments: the mono-nucleosome (~165 bp), di-nucleosome (~350 bp), and tri-nucleosome fragments (~565 bp). Although, each sample is different, and may display anywhere from one to six or more fragment sizes.

Continual improvements to NGS technologies have enabled the use of information-rich cell-free DNA (cfDNA) as a marker for liquid biopsies. Efficient and non-invasive screens for cancer, fetal health, and various genetic diseases are in development that are based upon the presence or absence of specific cfDNA sequences.

Levels of cfDNA in healthy individuals are generally low; however, during pregnancy, illness, and intensive exercise or injury, the levels of cfDNA increase. The ability to detect cfDNA has translated into non-invasive, alternative approaches for the detection of tumor mutations in cancer management, markers in the assessment of pathophysiological conditions, and screening tests for fetal abnormalities.

Genomic DNA (gDNA) has specific epigenetic patterns of methlylation or nucleosome positioning of the DNA that is specific to certain cell types. These specific patterns are retained in cfDNA and have been shown to be able to distinguish the tissue of origin. Epigenetic alterations such as differential methylation and nucleosome positioning have also been detected in diseased state cfDNA, allowing for the possibility of a blood-based biomarker for tumor development.

Analysis of cfDNA, such as mutation analysis and epigenetic alterations, is dependent upon next-generation sequencing (NGS) technologies. As such, cfDNA samples must undergo rigorous quality control analysis. The Fragment Analyzer™ Automated CE System, Fragment Analyzer INFINITY® Automated CE System, FEMTO Pulse™ Automated Pulsed-Field CE Instrument from Advanced Analytical Technologies, Inc. (AATI), with the associated reagent kits, provides a complete quality control solution for cfDNA samples.

Alternative methods (Download Application Note: "Quality Analysis of cfDNA with the Fragment Analyzer in Comparison with Another Electrophoresis System") of nucleic acid analysis lack the requisite detection sensitivity and separation resolution to accurately and reliably simultaneously quantify, size, and qualify cfDNA samples for use in downstream applications. Further analysis of cfDNA mutations and epigenetics requires NGS. Successful sequencing depends on multiple quality control checkpoints to provide high quality NGS libraries for sequencing. Quality control checkpoints provided guidance before library preparation, optimizes project outcomes, maximizes sample recovery, and overall provides reliable sequencing results faster and economically. QC analysis reduces the chance that subpar samples are carried through the entire process wasting time and money.

AATI provides several solutions for cfDNA quality control analysis. Several reagent kits are available for the Fragment Analyzer, Fragment Analyzer INFINITY, and FEMTO Pulse that provide the requisite sensitivity and resolution to reliably quantify, size, and qualify cfDNA fragments.

DNF-474 High Sensitivity NGS Fragment Analysis Kit

The DNF-474 High Sensitivity NGS Fragment Analysis Kit (HS NGS Fragment Kit) offers high resolution separation of the three cfDNA fragments with accurate and reliable sizing and quantification. Sizing helps to identify which cell and tissue type the cfDNA is released from and what type of cell death occurred. This reagent kit covers a size range larger than the cfDNA fragments allowing for separation from any fragmented DNA. Complete separation from extraneous DNA allows for accurate quantification which is essential when cfDNA levels are utilized as a screening tool for diseased states.

Some cfDNA extraction reagent kits utilize carrier RNA. Carrier RNA is used to help improve the isolation of cfDNA in some samples. The carrier RNA can be detected with the HS NGS Fragment Kit (Download Application Note: "Separations of cfDNA with DNF-474 by the Fragment Analyzer Short and Ultra-Short Array") and separates with the di-nucleosome cfDNA peak. When using this reagent kit, tt is recommended to avoid cfDNA extraction with carrier RNA for complete separation of all three cfDNA peaks, yielding reliable sizing and quantification data with the HS NGS Fragment Kit.

Figure 1. cfDNA with fragmented DNA separated with the DNF-474 High Sensitivity NGS Fragment Analysis Kit. Capillary electrophoresis performed on a Fragment Analyzer Automated CE System equipped with a short capillary array (33-55).

DNF-477 High Sensitivity Small Fragment Analysis Kit

Figure 2. cfDNA separated with the DNF-477 High Sensitivity Small Fragment Analysis Kit. Capillary electrophoresis performed on a Fragment Analyzer Automated CE System equipped with a short capillary array (33-55).

The DNF-477 High Sensitivity Small Fragment Analysis Kit (HS Small Fragment Kit) confidently sizes and quantifies the mono-, di-, and tri-nucleosome cfDNA fragments with high separation resolution. The reagent kit's small sizing range from 50 bp to 1,500 bp is perfect for reliable sizing and quantification of the small cfDNA fragments. The small sizing range of the HS Small Fragment Kit does not allow for complete separation of the tri-nucleosome cfDNA fragment from smaller fragmented gDNA. Incomplete gDNA separation (Download Application Note: "Accurate QC Analysis of cfDNA with the Fragment Analyzer") can affect the total cfDNA concentration and possibly sizing of the tri-nucleosome fragment. Carrier RNA occasionally used in cfDNA extraction does not interfere with cfDNA analysis on the HS Small Fragment Kit allowing for accurate quantification of the total cfDNA sample.

DNF-464 High Sensitivity Large Fragment 50 Kb Analysis Kit

The DNF-464 High Sensitivity Large Fragment 50 Kb Analysis Kit (HS Large Fragment Kit) offers complete separation with accurate and reliable sizing and quantification of all three common cfDNA fragments (Download Application Note: "Accurate QC Analysis of cfDNA with the Fragment Analyzer"), with the largest sizing range, 75 bp through 60,000 bp, of the three Fragment Analyzer reagent kits. An advantage of the wide sizing range is complete separation of fragmented DNA from the cfDNA fragments allowing for accurate quantification of the total cfDNA sample. The HS Large Fragment Kit also allows visualization of any possible cfDNA fragments larger than the tri-nucleosome fragment. Carrier RNA occasionally used in cfDNA extraction does not interfere with cfDNA analysis on the HS Large Fragment Kit, thus reliable quantification and sizing is obtained. The HS Large Fragment Kit is the recommended kit for analysis of cfDNA if gDNA contamination is a concern.

Figure 3. cfDNA with fragmented DNA separated with the DNF-464 High Sensitivity Large Fragment 50 Kb Analysis Kit. Capillary electrophoresis performed on a Fragment Analyzer Automated CE System equipped with a short capillary array (33-55).

DNF-493 High Sensitivity Large Fragment Analysis Kit

Figure 4. cfDNA separated with the DNF-493 High Sensitivity Large Fragment Analysis Kit. Capillary electrophoresis performed on a Fragment Analyzer Automated CE System equipped with a short capillary array (33-55).

The DNF-493 High Sensitivity Large Fragment Analysis Kit (DNF-493 HS Large Fragment Kit) offers complete separation with accurate and reliable sizing and quantification of all three common cfDNA fragments. The sizing range of 75 bp - 20,000 bp permits the complete separation of fragmented DNA from the cfDNA fragments, allowing for accurate quantification of the total cfDNA sample. The DNF-493 HS Large Fragment Kit offers separation and visualization of any cfDNA fragments larger than the tri-nucleosome fragment. The DNF-493 HS Large Fragment Kit is a reliable option for the analysis of cfDNA in the presence of larger gDNA.

FP-1101 Ultra-Sensitivity NGS Analysis Kit

The FP-1101 Ultra-Sensitivity NGS Analysis Kit (US NGS Kit) for the FEMTO Pulse offers separation of extremely low smear levels of 5 pg/µL – 250 pg/µL. Conservation of a sample is an asset for precious and low concentration samples. The US NGS Kit offers complete separation of the three cfDNA peaks (Figure 5 and 6) resulting in precise and dependable sizing and quantification. The reagent kit covers a sizing range, 100 bp – 6,000 bp, that enables separation of the cfDNA and larger DNA fragments. Complete separation of the cfDNA from larger DNA fragments enables accurate quantification required for screening tools based on comparison of cfDNA levels.

Figure 5. cfDNA #1 (35 pg/µL) separated with the FP-1101 Ultra Sensitivity NGS Analysis Kit. Capillary electrophoresis performed on a FEMTO Pulse equipped with an ultra-short capillary array (22-40).

Figure 6. cfDNA #2 (15 pg/µL) separated with the FP-1101 Ultra Sensitivity NGS Analysis Kit. Capillary electrophoresis performed on a FEMTO Pulse equipped with an ultra-short capillary array (22-40).

Cell-free DNA (cfDNA) Analysis Kits

The parallel capillary electrophoresis instruments developed by AATI provide accurate and versatile analysis of DNA and RNA samples. The Fragment Analyzer Automated CE System, the Fragment Analyzer INFINITY Automated CE System, and the FEMTO Pulse Automated Pulsed-Field CE Instrument provide variable throughput, high sensitivity detection, and a broad sizing range that exceed the base requirements for reliable nucleic acid QC in NGS workflows of any type.

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