This series of articles mentions the development in plant genomics and specifically the gDNA isolation. The challenges of the grinding, the lysis, the variety of crops and the automation possibilities are to be found in the previous articles.
This final part of this series describes the implementation of the CleanNA Clean Plant PK kit in an automated setting. The article will be based upon a case-study as performed in a Dutch plant breeding company. The process is implemented in the current automation setting of the customer. The quality is checked by using a High-Resolution Melt curve (HRM) application.
The goal of the project
The goal of this project is to automate the Clean Plant PK kit on the current liquid handlers as used by the customer. The liquid handlers can process multiple plates in parallel and are connected to the storage to enable an increased amount of plates per batch.
All programming and optimization is done by GC biotech. The results are obtained by the customer and compared to their current protocol. The goal is to obtain equal or better results for three different crop types with minimum adaptations to the protocol.
Adaptations to the standard protocol
To overcome the challenges as mentioned in the previous article, the Clean Plant PK kit is adaptable, scalable and uses some specific steps.
The Lysis buffer
Grinding and lysis is a challenge when processing plant material. The grinding during this project is performed separately from the lysis. The reason for this decision is that the amount of lysis buffer used per sample is varying per crop type. By varying the volume of the lysis buffer per crop, the following isolation results in an equal yield independent on the crop type.
The lysis buffer itself is based upon the most common home brewed method. It is Cetyl Trimethyl Ammonium Bromide (CTAB) based. This enables the use of the same lysis method for an extremely wide variety of crop types.
Downscaling the process
As the standard protocol is designed for higher input amounts, it uses higher volumes of buffers and reagents. The current version of the Clean Plant PK kit is suitable for downscaling and will therefor result in a lower price per sample.
During the optimization in this project, the entire kit has been downscaled by a factor 5, with an exception in the Lysis buffer. As mentioned, the lysis buffer is optimized per crop type and is delivered as a separate product next to the other components of the Clean Plant PK kit.
With the use of the adapted Clean Plant PK kit in the customer’s workflow, three different crop types have been used for validation purposes using HRM as a downstream application. After isolation, the yield and purity have been determined using a spectrometer. The obtained results have been compared to the current validated method. Using HRM, the melt curve analysis has been performed and compared to the current validated method.
Yield and purity
Table 1 shows the yield and purity obtained from the same 96 samples on one of the three crops. As seen, the typical yield is slightly higher compared to the current method. The purity of the DNA is based upon the 260/230 and 260/280 ratios and are equal for both methods.
Table 1: Yield and purity for crop 1
|Sample type||Average Conc (ng/µL)||Total Yield (µg)||SD||Highest conc (ng/μL)||OD 260/280||OD 260/230|
|Current method||Crop 1||17.58||2.19||1.869||21.75||1.64||2.92|
The higher yield and/or transfer of some cell debris after lysis made a difference in the PCR used for the HRM analysis. This has been solved by slightly varying the volume of lysis buffer in the initial reaction setup. By variation of the lysis buffer volume, the total process of DNA isolation is equal for all crop types used.
This yield and purity has been found for all three crop types used.
HRM melt curve analysis
The same 96 samples have been used for HRM analysis and have been compared to the current validated method. The results are shown in figure 1 (Clean Plant PK) and figure 2 (Current method). The HRM melt curve anaylsis showed some small optimizations need to be implemented on the lysis buffer volume used per crop. Slightly altering the lysis buffer volume used for each crop will eliminate the transfer of cell debris into the isolation plate. This further optimization will be done and tested by the customer.
Conclusion and summary
The three articles posted in this range have described the trends, challenges and the solutions provided by GC biotech in the field of plant genomics. The need for an increase in sample throughput, while maintaining a price level and flexibility can be solved by using the Clean Plant PK kit. The entire automation and optimization can be performed in close collaboration with the applications specialist of GC biotech, independent on the brand of liquid handling used. If there is a need for automation, GC biotech can provide a customer friendly automated process.
The results obtained with the Clean Plant PK kit have been equal or better compared to the current protocol as used by the customer up until today. The use of the downscaled protocol has resulted in a significant cost saving per sample, without making any concessions to the flexibility and throughput.