DNA from whole blood samples collected in PAXgene Blood DNA Tubes purified using the PAXgene Blood DNA Kit has an A260/A280 ratio of 1.7–1.9. Fragments range between 20–200 kb in size, with an average length of 50–150 kb (Figure 1). Average DNA yields are 150–500 µg, depending on the number of nucleated cells present in the blood sample.
Applications such as pharmacogenomic studies, gene expression studies, and SNP genotyping yield clinically important data and can require large amounts of purified DNA from individual subjects. Use of standardized methods for sample collection and nucleic acid isolation to produce high-quality DNA for such studies is becoming increasingly important for multi-center clinical trials and basic research. The purification process for the PAXgene Blood DNA Kit is performed in a single processing tube to minimize the risk of sample mix-up and cross-contamination. Together with the PAXgene Blood DNA Tube, which standardizes the collection and storage of whole blood at various temperatures, these two components of the PAXgene Blood DNA System comprise a simplified, integrated and standardized preanalytic workflow for blood samples used in DNA studies.
The procedure to purify DNA using the PAXgene Blood DNA Kit is shown in the flowchart (Figure 2). Briefly, blood samples collected in PAXgene Blood DNA Tubes are transferred to processing tubes (supplied already filled with cell lysis buffer), and the solution is mixed to lyse red and white blood cells. Cell nuclei and mitochondria are pelleted by centrifugation, washed, and resuspended in digestion buffer. Protein contaminants are removed by incubation with a protease. DNA is precipitated in isopropanol, washed in 70% ethanol, dried, and resuspended in resuspension buffer.
The resulting high-quality DNA can be used for demanding downstream applications, including:
- PCR, multiplex, long-range, and quantitative, real-time PCR
- Southern blotting
- SNP genotyping
- Next-generation sequencing