Over the last few decades, PCR, next generation sequencing and microarray technologies have taken blood-based research to a new level. Modern blood-based application range from DNA fingerprinting, whole genome sequencing, blood banking to liquid biopsy and many more. Regardless of the application, pure, intact, double stranded and highly concentrated DNA extraction from whole blood is an essential prerequisite to success in this area.
The technique you choose for whole blood DNA isolation impacts not only your results but also the ease of your molecular biology workflow. As with many other scientific techniques, choosing an extraction method can be a challenge in itself! Laboratories generally have their own preferred tried and tested protocol. Protocols can differ regarding setup and the exact chemical substances used to lyse cells and selectively isolate DNA. However, almost all of the available protocols for whole blood DNA isolation involve these common steps: cell lysis, removal of protein, other contaminants and RNA, and (finally) DNA recovery. This article will help you understand the different methods for genomic DNA isolation from whole blood.
DNA Extraction Protocol – Precipitation Chemistry
Whole blood DNA isolation using precipitation chemistry works by precipitating the DNA out of a lysate via high salt concentration and addition of alcohol (either ethanol or isopropyl alcohol). This method readily separates DNA from other biomolecules and organic compounds (e.g. macromolecules such as lipids and carbohydrates) that may interfere with downstream applications. This method will also separate DNA from any exogenously added chemical compounds that may be present in the blood (e.g. novel chemical entities in drug discovery programs). We can further subdivide the precipitation chemistry method into either one- or two-step lysis approaches.
In the two-step lysis approach (used in Puregene kits), the first step lyses red blood cells using detergents, such as sodium dodecyl sulfate (SDS) and Triton™ X-100. Then, a second step ensures lysis of white blood cells to release the cell nucleus, genomic DNA, and RNA (Figure 1a). Ribonuclease treatment (usually with RNAse A) then removes contaminating RNAs, leaving DNA and protein molecules behind in the supernatant.
The next step is to remove the protein using concentrated salts, which precipitate proteins from lysed cells. As the cation concentration increases due to the addition of the salts and free water becomes less available, protein solubility decreases and causes the proteins to be “salted out.” However, the DNA remains in the supernatant, which is collected.
In the one-step lysis approach (which is used in FlexiGene kits), the red and white blood cells are lysed in one step; this works via a detergent mix capable of lysing both cell types. This lysis step releases the nuclei and mitochondria from the white blood cells, which are collected via centrifugation, leaving the RNA in the supernatant (Figure 1b).
Like in the one-step (Puregene) approach, the next step is to remove contaminating proteins. Here, the white cell nuclei and mitochondria are treated with a denaturation buffer, which contains a chaotropic salt and a protease. This simultaneously denatures and digests the proteins in the cell nucleus and mitochondrion, leaving digested proteins and the genomic DNA in solution.
In both methods, DNA is recovered by alcohol precipitation and rehydrated using a Tris or Tris-EDTA buffer solution. Isolated DNA is stable at 4°C and -20°C for 10+ years.
The one-step lysis approach has a number of advantages over the traditional two-step lysis method:
- Most importantly for your DNA yield, it eliminates possible sample loss that can be incurred in a two-step procedure.
- No tube change is needed (i.e., for a manual procedure the risk of sample mix up is strongly reduced)
- It saves you time in the lab because there are fewer steps, and no overnight incubations or clean up steps. The two-step precipitation chemistry method, for example, can take up to 48 hours to get the DNA into solution.
- The one-step lysis approach is also friendly to your lab’s budget. It is less expensive because it doesn’t require extra enzymes, like RNase, or any additional equipment.
Extracting DNA Using Magnetic Beads
Magnetic bead capture is the newest method of extracting DNA. Whole blood DNA isolation using magnetic beads works by capturing DNA on magnetic beads coated with a matrix of silica for binding nucleic acids.
As with the precipitation chemistry methods, the whole blood cells first must be lysed using SDS or similar detergents. The next step involves mixture of the lysed cells and magnetic beads, allowing DNA to bind the beads.
Several rounds of washing in the presence of a magnetic field then separates the DNA captured on magnetic beads from other unbound cellular contaminants. Then, a low-salt buffer elutes the DNA from the beads.
Some studies, including a review by Carpi et al., indicate that magnetic beads can be the fastest method.1 However, this speed comes at a price—both monetarily and to your DNA yield and purity.2 The magnetic bead approach can result in up to 20% reduction to your DNA yield. Also, any magnetic bead carryover contaminates your DNA sample and interferes with downstream applications. Lastly, it is the most expensive method because it requires magnetic capture stands, plates, and magnetic beads.
Parting Words of Advice
No matter which method you choose, things can go wrong. Here are a few useful tips that you can incorporate to ensure high quality, high yield genomic DNA from whole blood for your downstream applications:
- Do a pilot experiment. Before embarking on a new technique with lots of samples, start out small. You will find out where the pitfalls are and you will be able to optimize your procedure to perfection before spending a lot of time and money on the real experiment.
- Use a DNA preservative. A number of DNA stabilizing reagents have appeared on the market in recent years. These are liquid reagents that you can add to your blood sample immediately after isolation to inhibit nuclease activity and potential contaminating microorganisms. This allows you to store your unprocessed blood for extended periods of time without having to worry about DNA degradation. Another advantage is that you can process all samples for an experiment together, thereby reducing a source of variation. Some of the commercially available products require removal of the stabilizer prior to downstream applications, so watch out for this!
- Quantity your DNA correctly. The presence of contaminants, degraded DNA and RNA in a genomic DNA preparation will influence your results. Unfortunately, you may not always catch these invaders if relying on one method of quantification. Therefore, it is wise to use a combination of spectrophotometry and agarose gel electrophoresis to both visualize and quantify genomic DNA, before embarking on time-consuming and costly downstream applications.
- Use the online community. There is a plethora of help available online from other scientists who may have solved the exact issue that is troubling you. ResearchGate, Protocol Online and LinkedIn groups are all worth checking out. Similarly, technical support from suppliers is another excellent resource for troubleshooting and general advice.
In the end…
Advances in biotechnology, cell biology, biochemistry and life sciences in general have really spoiled us for choice in the lab. Nowadays, it seems as though there is a kit for everything! However, as with almost all laboratory techniques from structural biology to molecular biophysics, each method has its own advantages and disadvantages. The protocol overviews and tips presented here should help you on your way in choosing a path for whole blood DNA extraction. The right technique for your research is probably the one that provides you with the best quality DNA easily and quickly.
- Carpi FM, Di Pietro F, Vincenzetti S, Mignini F, Napolioni V. (2011) Human DNA extraction methods: patents and applications. Recent Pat DNA Gene Seq. Apr;5(1):1-7.
- Chacon-Cortes D, Griffiths LR. (2014) Methods for extracting genomic DNA from whole blood samples: current perspectives. Journal of Biorepository Science for Applied Medicine. 2: 1–9.