One-Step Cell-Free DNA Purification Kit

Introduction

Cell-free DNA (or cfDNA) refers to all non-encapsulated double-stranded extracellular DNA fragments in the bloodstream and other bodily fluids, such as circulating tumor DNA (ctDNA), cell-free mitochondrial DNA (ccf mtDNA), and cell-free fetal DNA (cfDNA). This DNA is released into the bloodstream from various sources such as cancer cells, dead cells, and fetal cells. The extraction of cfDNA is important for various applications such as non-invasive prenatal testing, cancer diagnosis, and disease monitoring. Multiple cell-free DNA purification methods and numerous kits based on these methods and protocol variations exist. These various procedures and alterations result in a diverse set of cell-free DNA purification processes, which may affect cell-free DNA yield and purity. Due to the trace amount of circulating nucleic acid present in cell-free samples and the presence of other contaminants such as proteins, lipids, and RNA, scalable isolation of circulating nucleic acid from plasma, serum, and urine cfDNA recovery is challenging. The Cell-Free DNA Purification Kit provides a reliable and efficient method of purifying cfDNA from other contaminants.

The extraction of cfDNA is crucial for many applications in the life sciences, healthcare, and forensics. In prenatal testing, cfDNA extracted from the mother’s blood is used to determine the genetic makeup of the fetus, which helps detect any genetic disorders or chromosomal abnormalities. In cancer diagnosis, the presence of cfDNA in the blood is used to monitor the progression of the disease and to assess the effectiveness of treatments. In forensics, the extraction of cfDNA is used to identify individuals from blood samples found at crime scenes.

HygiaReagents Cell-Free DNA Purification Kit is designed to extract cfDNA from human plasma efficiently and sequentially. The kit uses our unique proprietary magnetic beads and an optimized buffer system. It can efficiently isolate circulating cell-free DNA (cfDNA) from 100 µL -10 ml (Typically yields about 25 nanograms of cfDNA) of human plasma using Cell-Free DNA BCT collecting tubes. The procedure employs mild lysis conditions, avoiding harsh conditions such as alkaline lysis and toxic chemicals for lysing cells to maintain DNA integrity and the time-consuming cleanup of organic solvent from the sample. Purified cfDNA has the highest integrity and can be used in various downstream applications such as qPCR, ddPCR, NGS analysis, etc. When combined with an automation system, multiple samples can be isolated simultaneously. Alternatively, samples can be processed manually using a magnetic stand.

Magnetic beads have several advantages over conventional approaches for isolating cfDNA. Beads more efficiently bind the DNA than glass fiber filters, resulting in higher and more consistent yields. Furthermore, because no filters or vacuum manifolds are employed, cellular particles cannot be clogged during the extraction process. This clogging problem is especially problematic in protein-rich, large-volume samples like plasma, often utilized in cfDNA research.

Workflow of cfDNA Purification Kit

The Cell-Free DNA Purification Kit uses a combination of magnetic beads and specific binding agents to purify cfDNA from other contaminants. The kit works by binding cfDNA to the magnetic beads and separating it from other contaminants such as proteins, lipids, and RNA. The purified cfDNA can then be used for downstream applications such as PCR, sequencing, and genotyping.

1. Add lysis buffer and proteinase K to the sample to lyse the bone and incubate at 65°C.

2. Add functional magnetic beads and vortex/pipette the beads with the sample to the DNA.

3. Wash the beads.

4. Separate the beads from the sample using a magnet.

5. Elute DNA from the beads.

Features and Advantages

● Reliable and efficient purification: The Cell-Free DNA Purification Kit provides a reliable and efficient method of purifying cfDNA, which is important for accurate results in downstream applications.

● High yield: The kit provides a high yield of cfDNA, which is essential for applications that require large amounts of DNA, such as sequencing and genotyping.

● The scalable format allows for higher and lower input volumes.

● Easy to use: The kit is easy to use and can be performed in a laboratory setting with the minimal technical expertise required.

● Versatile: The kit can be used for various bodily fluids, including blood, plasma, and other bodily fluids, making it suitable for various applications.

● Flexible design that enables both manual and automatic cfDNA isolation

● Automation-ready, phenol-free extraction

● Input volume ranges from 100µL to 10 mL of plasma

● Elution quantities range from 15 µL to 50 µL