Plants are a crucial part of our diet and the entire ecosystem we inhabit. In fact, plants are such a vital aspect of our planet that they account for over a quarter of new species discovered yearly (1). With this rapid level of discovery, researchers are in constant need to understand the genetic makeup and diversity of plant species. Lysing plant material to expose the genetic material contained within samples during preparation, however, can be quite difficult due to a variety of factors with the thick polysaccharide cell wall being among the most obstructive.
Enzymatic lysis is often used to break down these cell walls and expose the genetic material within; however, these methods can be time consuming and ensuring complete breakdown of the cell wall is very difficult. In addition to the challenging nature of the plant cell; every variety of plant type will provide their own obstacles due to their unique genomic makeup and could require optimization tailored to each species.
Enzymatic lysis however, is not the only form of lysis that can be utilized. Physical disruption is a frequently utilized alternative with some common implementations including manual methods such as mortar and pestle as well as vortexing. While these manual methods are suitable and are often cited in protocols or publications, they require manual effort on the part of the researcher which can introduce variability, 2 as well as limiting throughput. Introducing semi-automated sample preparation can help solve this problem by delivering a consistent and repeatable processing step. One such method can be found in physical bead-based lysis.
Bead mill homogenization can lyse difficult samples with consistency that manual methods struggle to achieve. Consistency is not the only value provided by homogenization however, in addition to this consistency, semi-automated bead beating offers a significant time savings compared to manual methods allowing the user to process far more samples than would be possible with other lysing methods. The Omni Bead Ruptor™ 96 bead mill homogenizer provides researchers with a robust sample prep solution that meets high throughput needs. Utilizing this instrument, users can process up to 192 samples simultaneously with enough force to homogenize their toughest samples prior to downstream processes.
Plant tissue is utilized in numerous downstream applications including PCR, sequencing, and genotyping among others. These nucleic acid-based applications require the genetic material contained within the plant tissue to be purified prior to analysis. As with lysis, several nucleic acid isolation techniques are available to the researcher; with manual spin column-based, or magnetic bead-based extractions being common manual extraction methods for the researcher. These methods, however, are limited in a variety of ways with the inherent variability of manual handling and limited throughput being of the most concern in plant genomic applications. Automating the nucleic acid extraction process not only ensures consistency from sample to sample, but also allows the researcher to purify a larger number of samples in only a fraction of the time that would be required otherwise. The chemagic™ 360 instrument enables automated nucleic acid extraction and utilizes patented magnetic bead technology and automated reagent filling to carry out automated extraction of nucleic acid from as many as 96 samples in one run.
As plants require greater amounts of genetic data to fuel the needs of next generation data analysis, higher sample volumes will be required to meet the demands of the rigorous research requirements ahead. Modern techniques such as next generation sequencing or long read sequencing are not only allowing for a deeper analysis of a sample’s genetic material; but also allow for an exponentially higher number of samples to be examined at once. As such, high throughput workflows are becoming a necessity in today’s research laboratories. In this work, we demonstrate a high throughput plant DNA workflow utilizing the Omni Bead Ruptor 96 bead mill homogenizer and chemagic 360 automated nucleic acid extractor for end-to-end sample preparation and DNA isolation from a variety of staple crops yielding high quality DNA suitable for next generation sequencing.
Figure 1: Spread of corn radicle DNA concentrations (ng/μL)