cfDNA (Circulating Cell Free DNA )
cfDNA or Cell Free DNA is known to circulate in healthy and pathological conditions and is present in plasma, serum, cerebral spinal fluid, and saliva. Circulating Cell-Free DNA (cfDNA) is present in the human blood at varying levels, from 1 to 100 ng per ml of plasma. The size of Cell Free DNA is typically 100 bp to 500 bp in length (with most fragments being about 160-180 bp long), making traditional DNA extraction difficult. The key to successful analysis of cfDNA is to ensure efficient purification of smaller DNA fragments (100-500 bp). We used cfPure™ Cell Free DNA Extraction Kit1 produced by BioChain, which efficiently recovers these important small fragments. The cfPure™ employs a magnetic bead-based DNA extraction method, for the recovered cell free DNA, which performs identically in sensitive applications, such as Next Generation Sequencing, to current spin column based cfDNA kits on the market. The recovered DNA is suitable for a wide range of down-stream applications, such as bisulfite sequencing, NGS, and qPCR.
QC Analysis of cfDNA using Qsep100TM CGE Bio-Fragment Analyzer:
Qsep100TM system that utilizes Capillary Gel Electrophoresis as the core technology with pen-shaped gel-cartridge (N1) was used for separation and fluorescence detection of the Cell Free DNA.
The automated Qsep100 system provides high resolution, high detection sensitivity and cost effective solution of cfDNA analysis for the non-invasive detection of cancer and prenatal genetic testing.
BioChain’s cfPure™ Cell Free DNA Extraction Kit was used for rapid and efficient purification of circulating cell free DNA (cfDNA) and transferred to BiOptic’s Qsep100 for electrophoretic separation and fluorescence detection.
The cfPure™ system utilizes silica-coated paramagnetic particles to purify cell free DNA from less than 1 mL to greater than 10 mL of serum or plasma. The buffers were developed to ensure efficient recovery of 100 bp – 500 bp DNA fragments in order to maximize recovery of cell free DNA.
The cfDNA was separated and detected by utilizing the high detection sensitivity N1 gel-cartridge. DNA Ladder (pBR322 MspI Digest diluted 100X from the 10 ng/ml stock solution) was used in conjunction with cfDNA samples for accurate size calling and quantitation.
Sample injection at 8KV, 20 seconds
Separation at 8KV, 150 seconds
Results analyzed by BiOptic’s Q-AnalysisTM Software
Figure 1. Cell Free DNA (test sample #1) at 169 bp with concentration of 3.12 pg/μl
Figure 2. Cell Free DNA (test sample #2) at 181 bp with concentration of 3.82 pg/μl
Figure 3. Cell Free DNA (test sample #3) at 171 bp with concentration of 8.21 pg/μl
Figure 4. Cell Free DNA test results + DNA Ladder (transposed electropherograms)