The EpiQuik™ Circulating Cell-Free DNA (ccfDNA) Isolation Easy Kit utilizes magnetic beads based size-fractionation technology to isolate circulating cell-free DNA (ccfDNA) from plasma/serum samples in a simple and fast manner. The isolated ccfDNA can be directly used for real time-PCR and DNA library preparation suitable for next generation sequencing. The kit has the following advantages:
- Fast and straightforward procedure can be finished within 1 hour.
- Uses innovative magnetic bead based size-fractionation technology for isolation of ccfDNA from plasma/serum in a simple and convenient manner.
- The isolated DNA can be directly used for both qPCR and NGS DNA library preparation.
- Efficient removal of proteins, salts, nucleases, PCR inhibiting substances, and other impurities such as polysaccharides, polyphenols and lipids.
- Sensitive and efficient DNA capture enables successful isolation with high recovery (>80% of input mononucleosomal DNA), even when the quantities of starting material are limited (as low as 0.2 ml).
Genetic and epigenetic analysis of circulating cell-free DNA (ccfDNA) in plasma/serum or other body fluids provides unique opportunities for early detection of a wide range of clinical disorders such as cancer, autoimmune disease, infection and fetal disorders. It was demonstrated that ccfDNA of clinical importance occurs predominantly as fragments of approximately 170 bases from mononucleosomes with a smaller proportion as fragments of 360 bases from di-nucleosomes [1,2]. Such nucleosomal complexes are released into blood circulation during apoptotic cell death and will be increased under various pathological circumstances such as inflammation, pulmonary embolism, autoimmune disease, and cancer [3,4]. It is also shown that ccfDNA from nucleosomal complexes in serum and plasma is small size fragment DNA (170-500 bps) and using such ccfDNA for genetic or epigenetic analysis provides better and more accurate identification of physiological and pathological status .
Principle & Procedure
The EpiQuik™ Circulating Cell-Free DNA (ccfDNA) Isolation Easy Kit contains all components which have been optimized for the simple and rapid isolation of small size ccfDNA from plasma/serum. The circulating nucleosomal complexes are first digested and DNA is then enzymatically released. The ccfDNA is efficiently captured via size-fractionation magnetic beads (ccfDNA Capture Beads) by applying the beads to a magnetic field (EpiMag™ HT (96-Well) Magnetic Separator or similar). The captured ccfDNA is purified by simply washing the beads. The purified ccfDNA is then eluted from the beads for immediate use or storage.
Both fresh and frozen plasma/serum from various sources can be used. However, fresh plasma/serum will generally give higher DNA yields than frozen. The input volume of plasma/serum can be from 0.1-1 ml with the standard volume of 0.5 ml per sample. If serum sample is used, the serum should be prepared within 6 hours after blood draw, since lysis of peripheral blood lymphocytes may cause an artificial increase in the amount of DNA during serum separation.
1. Jahr S et al: Cancer Res. 2001, 61: 1659-1665
2. Suzuki N et al: Clin Chim Acta. 2008, 387: 55-58
3. Holdenrieder S et al: Crit Rev Clin Lab Sci. 2009, 46: 1-24
4. Schwarzenbach H et al: Nat Rev Cancer. 2011, 11: 426-437
5. Chan KCA et al: Clinical Chem. 2004, 50: 88-92
Fig. 1. Workflow of EpiQuik™ Circulating Cell-Free DNA (ccfDNA) Isolation Easy Kit.
Fig. 2. High recovery of ccfDNA: Different amounts of HeLa mononucleosomes were spiked into 0.5 ml of plasma then isolated using the EpiQuik™ Circulating Cell-Free DNA (ccfDNA) Isolation Easy Kit. The isolated DNA was fluorescently quantified.
Fig 3. High recovery of ccfDNA confirmed by bioanalyzer analysis: 500 ng of Hela polynucleosome (around 3000 bps) and 500 ng of mononucleosome (around 170 bps) were simultaneously spiked into 0.5 ml plasma and then isolated and size-selected.
Fig 4. Bioanalyzer trace of DNA library prepared from 20 ng of purified mononucleosomes isolated from plasma spiked with both mono- and poly-nucleosomes. Library peak size: 350 bps.