Developing a rapid method of DNA extraction from blood is crucial for effective research, medical diagnostics, and forensic investigation. Given the broad range of end-uses for blood extracts, sampling methods and downstream applications can vary substantially. Thus, selecting an extraction protocol capable of producing high quality DNA from a wide range of source material is critical.

The PDQeX Blood extraction system takes the multistep, time-consuming process of extracting PCR-ready DNA from whole blood, and turns it into a simple, single step method using the ZyGEM PDQeX2400. The one-step, closed-cartridge method both lyses the cell and purifies the DNA, maximising yield and minimising the risk of contamination. Cells are lysed using an aggressive thermophilic proteinase and the lysate purified as it flows through a matrix at the outlet to the PDQeX cartridge. DNA is free of cell debris and inhibitors.

Typically, DNA from a PDQeX extraction can be used for endpoint PCR, qPCR, and STR analysis. However, due to the final high temperature step of the PDQeX program, the DNA is largely single-stranded, and is therefore not able to be used in Next Generation Sequencing unless the library construction has a PCR step.

Typical Results

Liquid Blood Extractions

5 µl of whole blood from an EDTA BD Vacutainer® and fresh blood from a finger-prick was extracted using the prepGEM Blood PDQeX kit. 5 µl of the extract (total volume 100 µl) was used in a 20 µl qPCR reaction. The plots of the two samples are shown below.

 

 

 

 

 

 

BRCA 1, qPCR plots showing fresh blood (Black) and EDTA (Blue) with human male standard DNA (Red) at concentrations of: 10 ng/µl, 2 ng/µl, 0.4 ng/µl, 0.08 ng/µl.

We recommend using the PDQeX Blood kit for liquid blood, blood stored on cards and bloodstains. Blood extracts require the purification polymer integrated into in premium tubes that are supplied with the blood kit.

However, if you are using haem-resistant PCR or STR reagents for your analysis, it is possible to use PDQeX Standard cartridges for blood, and this may provide a lower-cost option – especially if your target DNA is at sufficient quantity that a dilution of the crude extract can be made.

The qPCR plot below compares 20 µl qPCR reaction with 5 µl of either an undiluted blood extract or 5 µl of a 1:10 dilution. The plots show the results obtained using both PDQeX Standard and PDQeX blood.

 

Blue = PDQeX blood Kit, extract undiluted (Efficiency: 102%)

Magenta = PDQeX blood Kit, extract diluted 1:10 (Efficiency: 94%)

Black = PDQeX Standard Kit, extract undiluted (Efficiency: < 50%)

Red = PDQeX blood Kit, extract diluted 1:10 (Efficiency = 92%)

Green = Standard curves (2 ng/µl, 0.25 ng/µl, 0.031 ng/µl, 0.0039 ng/µl)

 

If you need help and advice, please contact technical@zygem.com.

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