Trials and tribulations of a plant scientist

DNA extraction from plants can be a drawn-out process for the most scientists, cloning and automation provide only part of the solution

The life of a plant scientist can be particularly difficult when DNA isrequired to support their investigations. From harvesting through to purification, obtaining DNA can be a long, difficult and even dangerousprocess. With an increasing demand for genomic and genetic studies across a widerange of plant species there is an increasing need to reduce theinconvenience and cost involved in obtaining DNA to support this work. Steve Dodsworth, Nucleoplex Development Manager at Tepnel Life Sciences, reviews the inherent difficulties in DNA purification from plant material and how large scale studies are becoming more feasible with developments to overcome these problems.

When DNA is needed to support large scale plant studies the limitations of purification technology restrict the science itself. It can be challenging to extract DNA from just one plant but it’s far more difficult if you require DNA from hundreds or even thousands of specimens.

There are many scientists who have become familiar with the high throughput purification of plasmid DNA from bacteria. Bacterial growth medium contained within 96 well plates is inoculated with E.coli. clones from plated colonies or storage archives - there are even colony picking robots to increase the efficiency of this process. The clones are grown overnight in a rotary incubator and their plasmid DNA is then extracted using one of a plethora of effective kits with many automated platforms to support high throughput purification. How plant scientists would like the ease and efficiency of this process applied to their own field.

The first challenge for the plant scientist is to collect the plants for study. The excitement and adventure of collecting plant specimens from the field can be easily offset by the trips, stings and scrapes of negotiating rough terrain. Every year there are casualties sustained in the name of specimen collection for the prized quarry inevitably resides in some unwelcoming and hazardous situation. Once collected there is the question of storage and transport to the distant laboratory.

You can perhaps understand the desire for cultivation in a controlled glass house environment if this option is feasible but while the modern glass house offers safety and control, there are many factors which must be critically varied. Ventilation, illumination, humidity and temperature must be provided as part of an exacting discipline along with careful watering and the control of pests and diseases. This of course presumes that perhaps a hundred, a thousand or even more seeds are germinated and the seedlings planted with careful annotation to describe timing, location and identity. Automation and software are critical in the modern greenhouse but the need for manual labour has not been totally elliminated.

Assuming the desired plants are available a researcher must face up to the task of DNA preparation. A wide range of “home brew” methods can be found on the internet but most are time consuming and specific to a certain tissue from a certain plant. It is not unusual to take perhaps 2 days to prepare DNA from 48 samples and many a post-doc or technician will have to suffer a long and drawn out process to support a study of any substantial size. The study may be compromised if samples are lost, mixed up or the quality of the resulting DNA is poor due to a lapse in concentration during these extended procedures.

There are good kits available to prepare DNA from plant material. For example, Tepnel’s Nucleon Phytopure kit provides a robust method for small sample numbers with the advantage of large fragment sizes resulting from it’s solution phase chemistry. Companies such as Qiagen provide purification kits in a 96-well format kit to support higher throughput. These are typically based on vacuum manifolds that draw plant lysate onto a silica matrix for the binding of DNA. Whilst shearing forces reduce the resulting fragment size, the eluted DNA is still suitable for many downstream applications. A multi-channel pipette is an essential tool for use with such kits and care should be taken to push pipette tips on firmly and to make sure the correct sample row is accessed during reagent additions and liquid transfers. When using kits without a filtration step, the prospect of pipette tips blocking with plant debris is yet another potential pitfall for the unwary. Whilst competition will always drive prices down, the plastic goods in vacuum based kits are expensive which often means that suppliers cannot drop prices to adequately meet the needs of those with high throughput requirements.

Before the purification process can begin in earnest, whether using home brew or a commercial kit, tissues require mechanical disruption. Whilst grinding in a mortar and pestle over liquid nitrogen is effective it is exhausting and only suitable for a handful of samples. A godsend to the plant scientist is the automated grinder or mixer mill. Plant tissues are placed in tubes or deep well plates in the presence of ball bearings and the vessels sealed and placed on a mixer mill. The mixer mill violently shakes the samples with the ball bearings, a process which reduces most tissues to a powder if they have been dipped in liquid nitrogen shortly before grinding. Other methods use mechanical disruption in the presence of a lysis buffer at room temperature but this may be inadequate to thoroughly disrupt tougher plant tissues leading to a loss in yield, quality and reproducibility. In addition, the shearing forces can lead to a reduction in the length of the resulting DNA fragment. At the end of the process it is not unusual for researchers, short on cash, to spend more time recovering and (acid) washing the ball bearings for use in future grinding.

The 96-well format mixer mill has helped plant DNA purification step-up a gear but manual DNA purification techniques have been slow to keep pace. If manual methods cannot truly support high throughput plant DNA preparation then perhaps automation can provide an answer. Unfortunately such DNA purification methods are difficult to automate as the shattered plant material forms a particulate soup which can be impossible to pipette. Centrifugation provides only a partial solution as many plant tissues do not readily sediment blocking automated pipette heads that attempt to transfer the cleared lysate away from the debris for further purification. In addition, the automated method is only as good as the chemistry on which it relies and samples with elevated levels of polyphenolics, polysaccharides or oils may well overwhelm the chemistry resulting in contaminated DNA which will not perform in down stream applications.

Some of the X, Y, Z robotic systems on the market can be adapted to provide semi-automated methods for specific sample types but these may lie beyond budgetry constraints of small and medium size research groups. In addition, the flexibility provided by these systems does not usually enhance their ease of use and researchers may be put off by the prospect of extensive training or simply the reality of teaching the robot the movements required to support the purification process.

The Nucleoplex DNA purification system from Tepnel Life Sciences is a quantum leap for the plant scientist looking to ramp up their purification practices. The dedicated DNA preparation system is a compact bench-top instrument with pre-set protocols. Advances in lysis technology have resulted in a method which alleviates the need for liquid nitrogen and breaks down the toughest tissues in combination with a mixer mill. To support this approach, disposable ball bearings are provided in collection tubes which themselves are provided in a 96 format rack which can be loaded directly into the instrument. The size of the resulting DNA fragments is controlled by choosing one of three lysis protocols using the touch screen interface. In the Nucleoplex system, the ratio of the lysis solution to the neutralisation solution is the dominant factor in controlling tissue breakdown and DNA fragment size and not (perhaps surprisingly) the mechanical shearing forces of the ball bearings during milling. The combination of chemical attack, enzymatic digestion and mechanical disruption destroys the plant structure forming a fine suspension for further processing liberating individual cells for lysis, thus maximising yield.

A removable filter tip is present on each pipette tip to alleviate pipette blockage. The cleared lysate is then bound to magnetic beads which do not have a silica surface commonly used in manual purification kits. The advantage for the plant scientist is that the binding characteristics of the bead surface support the provision of highly pure DNA upon elution. This robust and novel approach allows the system to effectively deal with both ‘easy’ and ‘difficult’ sample types using the same reagent set. With a training time of less than 15 minutes, a set up time of 5 minutes and a throughput of 384 samples per day, the system allows plant scientists the freedom to concentrate on their science rather than the challenge of DNA extraction.

The importance of plants in our food supply has never truly been reflected in research funding but with the availability of genome sequences and a move to functional genomics, plant science is being carried forward at pace. The ease of high throughput purification which is now taken for granted by “plasmid preppers” but was once a distant prospect in plant science is now very much a reality available to even small research labs as costs decrease and convenience increases.

By Steve Dodsworth, Nucleoplex Development Manager, Tepnel Life Sciences