Cell Free DNA Extraction kit – BIO4V-TECHNOLOGIES SAS

Aperçu

Les kits d’extraction d’ADN « cfPure® Cell Free” sont conçus pour une purification rapide et efficace des ADN circulants libres de cellules (cfDNA). Le système utilise des particules magnétiques recouvertes de silice pour purifier ces ADN libres circulants depuis 1 ml jusqu’à plus de 10 ml de sérum ou de plasma. Les tampons ont été développé pour assurer l’efficacité de l’extraction de fragments d’ADN de 100 paires de bases à 500 paires de bases afin de maximiser la récupération d’ADN libres circulants. L’ADN récupéré convient à une large gamme d’applications en aval comme le séquençage NGS, les qPCR et les ddPCR.

Applications:

Extrait un ADN libre circulant de haute qualité à partir du plasma, sérum, et de l’urine pour le séquençage de nouvelle génération ( NGS), la PCR, la qPCR, et la ddPCR et bien d’autres applications.

Advantages and Features:

• Versatile: Extraction from <1mL to >10 mL serum or plasma – in single tubes or 24 to 96-well plates
• Automation Friendly: On both the KingFisher™ and liquid handling instruments, with extractions taking approximately 50 minutes
• Time Efficient: Manual extraction protocol takes less than 75 minutes
• Cost Effective: Competitive price <3€ per extraction from 1 mL plasma (List price for 250 mL kit)
• Elution volumes: Range from 15 µL to 50 µL

cfDNA Kit Yields Reproducible Results

Plasma ID	Total DNA Recovered (ng)
A	7.00
A	7.05
B	32.45
B	33.50
C	26.80
C	27.70
D	10.90
D	10.90

Table 1. To assess reproducibility, DNA was extracted from 1 mL of plasma from 4 different human donors in duplicate using the cfPure® cell free DNA extraction kit.

Efficient Recovery of cfDNA Relative to Competition

Extraction Kit	Sample ID	cfDNA recovered per ml of plasma (ng)	115 nt ALU sequence cfDNA per ml of plasma (ng) (by PCR)
Competitor T	1	4.99	1.97
BioChain’s cfPure	1	6.35	2.53
Competitor T	2	6.18	3.21
BioChain’s cfPure	2	8.28	3.93
Competitor T	3	6.93	3.79
BioChain’s cfPure	3	7.50	3.87

Table 2. cfDNA was purified from 2 mL of plasma from three different donors by either the cfPure® Cell Free DNA Extraction Kit or by a competitor’s magnetic bead-based cell free DNA extraction kit. DNA recovery was assessed either by a fluorescent method or by qPCR using primers complimentary to ALU sequence DNA.

Figure 1. cfPure® Cell Free DNA Kit was used to isolate DNA from 5 ml of healthy human plasma. 62 ng of cfDNA (150-200 bp fragments) were recovered with low amounts of gDNA contamination.

**Figure 2.** cfDNA was extracted from the plasma of two human donors using various sample volumes(.2, .5, 1, 5, 10ml). The cfDNA yield was then quantitated by Qubit High Sensitivity DNA assay. The yields were plotted against the starting sample volumes and the R² values of their trend lines were calculated.

cfDNA Validated with NGS by End Users

cfDNA extracted by cfPure^® cfDNA Extraction kit is excellent for downstream applications such as NGS. In a third party study cfDNA was extracted from 10 ml’s of human plasma from 4 donors using BioChain’s cfPure^® (B), Competitor T’s(T), and Competitor Q’s (Q, column-based) cfDNA extraction kits. Libraries were then constructed for each sample , and whole genome sequencing was performed . From the data analysis, three extraction methods did not show significant performance difference across the board: Insert size, Mapping rate, Whole genome coverage, Substitution rates and GC bias.

**Figure 3.** Number of reads against the length of the inserts. Each curve represents average of 4 samples from the same extraction method.

**Figure 4.** The percentage of coverage or depth in a GC bin is calculated by dividing the number of reads of a particular GC content by the mean number of reads of all GC bins. A number of 1 represents mean coverage, a number less than 1 represents lower than mean coverage while a number greater than 1 represents higher than mean coverage.

Automation Ready

cfPure® Cell Free DNA extraction kit is applicable on both the KingFisher™ and liquid handling instruments. With the KingFisher™ Flex and BioChain’s cfPure® Cell Free DNA Extraction kit can extract up to 96 one ml samples in 50 minutes, or 24 five ml samples in one hour.

**Figure 5.** cfDNA was purified from 4 mLs of EDTA plasma, using the KingFisher Flex automated purification system and either BioChain’s cfPure® system (cfPure KingFisher), or a competitor’s kit (Competitor T KingFisher). Both automated extraction methods were compared with extractions done manually with cfPure® (cfPure Manual). cfDNA recovery was assessed by qPCR, using primers complementary to ALU repeat sequences.

Specifications

Sample Type (General):	Liquid Samples (e.g. Serum), dsDNA
High Throughput Compatibility:	Automated Protocols
Downstream Application:	Next-Generation Sequencing, PCR, Real-Time PCR
Product Line:	cfPure®
Product Size:	1 kit
Shipping Condition:	Room Temperature

Kit Contents

K5011610	K5011625	K5011625MA
2 x 1.33 mL cfPure Cell Free DNA Magnetic Beads	5 x 1.33 mL cfPure Cell Free DNA Magnetic Beads	5 x 1.33 mL cfPure Cell Free DNA Magnetic Beads
1 x 115 mL cfPure Cell Free DNA Lysis/Binding Solution	3 x 95 mL cfPure Cell Free DNA Lysis/Binding Solution	1 x 95 mL cfPure Cell Free DNA Lysis/Binding Solution
2 x 55 mL cfPure Cell Free DNA Wash Solution	5 x 55 mL cfPure Cell Free DNA Wash Solution	1 x 65 mL cfPure Cell Free DNA Wash Solution
1 x 6 mL cfPure Cell Free DNA Elution Solution	1 x 15 mL cfPure Cell Free DNA Elution Solution	1 x 15 mL cfPure Cell Free DNA Elution Solution

Citations

Feasibility of blood testing combined with PET-CT imaging to screen for cancer and guide intervention

Lennon AM, Buchanan AH, Kinde I, et al.

[published online April 28, 2020]. Science. doi: 10.1126/science.abb9601

Assessing aneuploidy with repetitive element sequencing.

Douville C, Cohen JD, Ptak J, Popoli M, Schaefer J, Silliman N, Dobbyn L, Schoen RE, Tie J, Gibbs P, Goggins M, Wolfgang CL, Wang TL, Shih IM, Karchin R, Lennon AM, Hruban RH, Tomasetti C, Bettegowda C, Kinzler KW, Papadopoulos N, Vogelstein B.

Proc Natl Acad Sci U S A. 2020 Mar 3;117(9):4858-4863. doi: 10.1073/pnas.1910041117. Epub 2020 Feb 19. PMID: 32075918

Cell-free DNA in cancer: current insights.

Fettke H, Kwan EM, Azad AA.

Cell Oncol (Dordr). 2019 Feb;42(1):13-28. doi: 10.1007/s13402-018-0413-5. Epub 2018 Oct 26. Review.PMID: 30367445