A new technical article in The Plant Journal describes a method developed by the authors to ascertain the number of transgenes inserted into a variety of important crops.
Knowing the number of genes successfully inserted into an organism as a result of a transformation event is useful information. Although there are simpler methods to determine whether a plant has been successfully transformed, being able to determine whether there is one or more transgenes present assists understanding the effect of one gene being inserted on plant phenotype and its segregation through following generations. It also allows researchers to eliminate the possibility of multiple copies of the gene being inserted and resulting in silencing of the gene.
The researchers outline the current problem with attempting to determine the number of genes inserted into an organism. Southern blot analysis is used to confirm the insertion of target genes, but even the most skillful analysis can struggle to identify when multiple transformations have occurred in the same segment of the dissected genome. Further, the analysis is expensive, labour intensive and requires the use of radioactive materials, in turn requiring special permits and handling procedures.
Quantitative Polymerase Chain Reaction, although rapid, isn’t able to provide the accurate measurement data obtained from Southern blot analysis and distinguishing between one and two transgenes is difficult even with extensive optimisation.
A More Accurate, Faster Method
The researchers made use of a newer PCR method, droplet digital PCR. Here is a short video covering some of the basics, but there are more detailed videos covering the technology:
For further reading, here is a 2012 Nature review of the technology that explains the process and its advantages really well as well as pointing out the differences with qPCR, and here is another from the IDT website.
The importance of this method lies in the use of the small droplets into which the reaction mixture is divided and in which the PCR amplification takes place. The division of the fractionated genome into the droplets results in either one or no DNA segments being contained within any particular droplet. With each droplet containing primers for the gene of interest for PCR amplification and a labeled probe for identification, subsequent analysis of the amplified droplets can detect the number of droplets containing the sequence of interest.
Figure from Nature review of dPCR illustrating the division of DNA between droplets and subsequent amplification.
Using the number of positive and negative reactions combined with the volume of the sample, the number droplets used and a Poisson statistical analysis to determine the likelihood of more than one segment of DNA being contained within a single droplet, a precise determination of the number of times the gene of interest is present in the genome can be performed. It is even possible to determine hemizygous or homozygous presence of the gene.
In order to determine the number of transgenes inserted into a genome, a known single-copy gene within the same genome must be selected for identification and comparison.
Making it Work for Plants
The purpose of the research was to optimise the use of droplet digital PCR for use in a number important crops, particularly rice, citrus, potato, tomato, maize and wheat, and to test the results of their protocols against Southern blot analysis results of the same plants for validation. The result, they hope, is to provide a cheaper, more efficient method to determine transgene number in plants that can then be used confidently in further studies.
To perform the study, the researchers picked common transgenic target sequences and single copy genes in each of the subjects to serve as a reference and ran a duplex analysis of each of the transformed food crops.
For each of the 6 subjects, the paper identifies the both the transgene and the reference gene used, whether the analysis was conducted in the transformed adult or progeny plant and how successful the analysis was in determining transgene copy number when compared with the Southern blot analysis.
Even with the varying sizes of the genomes tested, the results of the digital droplet PCR in each crop were comparable to the Southern blot analysis, providing confidence that the method could be used in transgenic research.
Some things to consider
Although the assessments were successful, the researchers note a few observations they made to assist anyone else using the technique, including:
- there is the possibility of obtaining ‘rainy’ droplets, where the droplet was found to be positive for the gene of interest but for which the signal wasn’t as strong as it was for the majority of the positive droplets. Despite the observation, the ‘rainy’ droplets didn’t appear to affect the result;
- plants with larger genomes require a larger quantity of genomic DNA for analysis in order to obtain an accurate analysis;
- sing either T0 plants or plants of known zygosity is important for an accurate analysis of transgene copy number using a single reaction;
- quality, accurately measured DNA must be used for analysis to avoid poor quality droplets;
- there is a range of optimal amounts of genomic DNA to be used dependent on the genome size in order that the confidence levels derived from the Poisson statistics are high enough that the results can be relied upon;
- fractionation methods chosen for use are important. Complete digestion to allow for random segregation in the droplets of fragments is required;
- if too few droplets are created the sample has been inadequately partitioned and results should be looked at closely; and
- copy numbers that are midway between integers indicate a problem with the reaction.
The paper outlines a method that other researchers can use to cheaply and accurately determine the number of transformation events in crop plants and should contribute to the efficiency of research in transgenic laboratories. The amount of reaction mixture, the primers and reporters used and the plant populations that the DNA are cultivated from can all be adjusted from the methods used in the paper and will hopefully lead to usable protocols in numerous plants.
* Feature image credit: Integrated DNA Technologies – http://www.idtdna.com/pages/decoded/decoded-articles/core-concepts/decoded/2013/10/21/digital-pcr-(dpcr)-what-is-it-and-why-use-it-