One of the most time-consuming bottlenecks in molecular biology experiments is efficient grinding, homogenization, and lysis of starting materials. This is especially important in plant research, soil, and environmental sciences, as well as for more common biomedical research areas, including extraction of DNA, RNA, and proteins from microbiological and solid tissue samples. For example, it is estimated, that in plant biology up to 20% of a lab technician’s time is spent on sample lysis.
Regardless of the nature of the research, it is essential that samples are lysed quantitatively. Equally important is that the simultaneously released macromolecules are functional and unaltered. In order to preserve fragile structures, it is often necessary to perform lysis and homogenization rapidly and at lower temperatures.
Sample Lysing Technologies
Most researchers today are using chemical, enzymatic, or low-tech mechanical sample homogenization methods. Problems surrounding these methods include inconsistent results due to operator and sample variability, the low-throughput bottleneck of a single sample-processing operation, and long processing times for hard, solid materials like plant tissues or bones.
The most common sample lysis method is grinding samples with a mortar and pestle, either at room temperature or cryogenically using liquid nitrogen. A mortar and pestle is largely unsuitable for low yield molecules and for ultrasensitive downstream detection techniques because it is virtually impossible to assure the absence of sample-to-sample cross contamination.
Other popular methods of sample preparation include ultrasonication, wherein the propagating ultrasound waves shear samples, and handheld, rotor/stator type homogenizers, both of which are limited to soft or suspended samples.
In addition to classical mechanical processes, chemical and enzymatic digestion or lysis are mainly used for cell culture and soft tissue. Examples include proteinase K or Zymolyase enzymatic lysis of mammalian tissue and yeast, however the process is time consuming, often up to 24 hours, and requires incubation at elevated temperatures. Problems arise if the molecules of interest are thermally unstable, prone to enzymatic degradation, or they are present in low abundance.
Mechanical sample lysis can be achieved in multiple ways, however, the nature of the forces acting on the sample can be one of three: cascade impaction, shearing via shear forces, and shearing by fluid vortexing. In cascade impaction, which is essentially the “hammering” of a sample with another object, the direct impact of compression induces cracks and breaking up of the sample. An example is bead beating, where the samples are exposed to the simultaneous impaction of high-density inert ceramic or metallic beads.
Shear forces work by imposing slicing motions on a sample to shred it apart, and can be applied mechanically like with safety razors or by fluid mechanics like the shear flow in a French press or in vortexer. Due to the complexity of 3-D random motion and complex velocity fields in a vortexer, the vortexing process, although strictly speaking is part of shearing, is also considered a separate milling process.