Introduction

Fluorescent probe technology allows for rapid genotypic identification. This information can be used for identification of allele frequency followed by linking the genotype to a phenotype. Genotyping is extensively used in genetics research like crop science, cancer research and hereditary studies. As more and more genomes are published every day and the genes within the genomes are narrowed down and understood, genotyping is becoming a very powerful tool to rapidly identify beneficial traits and dispose of linkage drag in crop science, determine zygosity, detect hereditary diseases and also to identify various cancer types. There are multiple ways to tackle genotyping experiments; be it by sequencing or using PCR genotyping by hydrolyzing probes and FRET assays. The intention of this application note is to focus on using hydrolyzing probes and FRET assays. 

Application purpose

SNP markers are available in many different formats; SSR, AFLP, CAPS, probes and FRET just to name a few. Most of these markers are difficult to automate, like the traditional gel-based markers. Hydrolyzing probes and FRET assays are end-point genotyping techniques meaning that they can be run in a traditional PCR instrument. After amplification the plates can be measured using a plate reader with distinct filters for excitation and emission capturing the end point fluorescence. Traditional PCR instruments are more cost efficient compared to qPCR instruments and offer greater flexibility for increasing throughput. Using single PCR instruments has another advantage; to allow for utilizing different PCR protocols differing in annealing/extension times as well as running different assays, even using the platform for standard PCR reactions to be later placed on gel.  
Key features
  • Scalability: To fit a higher throughput more PCR instruments can be added.
  • Flexibility: The individual PCR instrument can execute different protocols.
  • Walk away: High input and output options offer a long hands-off time.
  • Precise: Avoid human pipetting errors.

Why should you choose this application

Genotyping can be approached in many different ways. The application described above offers medium throughput of about 20.000 datapoints per day with high flexibility. Due to the single operating PCR instruments and flexible PlateButler software different protocols can be run on different PCR instruments thereby tailoring to a demanding lab environment.