How does a realtime PCR allelic discrimination also called Genotyping work?
What are the different realtime PCR probe formats to do allelic discrimination?
What are the pros and cons of each?
Allelic discrimination are methods to discriminate between 2 or more different alleles. For example in a [G/A] SNP there are 3 genotypes:
– Allele1: “G” in a diploid homozygote: GG. Maybe Wt (wild type)
– Allele3: “A” in a diploid homozygote: AA. Maybe Mut (mutant)
– And Genotype2: in a diploid heterozygote: GA
I did not mix up the allele/genotype 2&3 this is deliberate. There are 2 different alleles for this G/A SNP and 3 different genotypes and the common nomenclature is to call the heterozygote: “2”
How does a realtime PCR allelic discrimination also called Genotyping work?
PCR is PCR to amplify the SNP or Indel (insertion-deletion) sequence. Then in the realtime PCR there are several different probe or DNA intercalating dye systems to distinguish between the different genotypes and or alleles.
What are the different Realtime PCR probe formats to do allelic discrimination?
There are several different methods to do realtime PCR. All methods have advantages and disadvantages.
The most common and popular (but not necessarily the best) is Taqman Genotyping or Taqman allelic discrimination.
https://www.applied-maths.com/applications/taqman-based-snp-genotyping
At the end of the PCR reaction, the fluorescent signal for the two reporter dyes is measured. The ratio of the signals will be indicative for the genotype of the sample (see figure).
There are many advantages: Simple, robust, all realtime PCR popular instruments have SW analysis.
The main disadvantage is that you need for each SNP 2 double labeled probes, one probe for each allele labeled with both a Fluorophore and a Quencher.
There is a common and popular improvement/ or development for this Taqman method called KASP (Kompetitive Allele Specific PCR).
https://www.biosearchtech.com/support/education/kasp-genotyping-reagents/how-does-kasp-work
This method is very similar to the Taqman method , the main and only advantage over Taqman is costs. To my personal experience, less robust. And also requires the KASP vendor to design the primers and probes.
HRM High Resolution Melting
https://en.wikipedia.org/wiki/High_Resolution_Melt
HRM analysis is performed on double stranded DNA samples. Typically the user will use polymerase chain reaction (PCR) prior to HRM analysis to amplify the DNA region in which their mutation of interest lies. In the sample tube there are now many copies of the DNA region of interest. This region that is amplified is known as the amplicon. After the PCR process the HRM analysis begins. The process is simply a precise warming of the amplicon DNA from around 50 ˚C up to around 95 ˚C. At some point during this process, the melting temperature of the amplicon is reached and the two strands of DNA separate or “melt” apart.
The main advantage is that it cost less and you don’t need to use sequence specific labeled probes.
Another big big advantage is that potentially you can detect any small changes/mutations in the amplified DNA (also known as amplicon).
The disadvantages is that it works poor on poor extracted DNA. And to my personal experience far far less robust as a diagnostic allelic discrimination method in compare to other probe based systems.
SimpleProbesTM (this is a Roche trade mark)
https://lifescience.roche.com/documents/Assay-Formats-for-Use-in-Real-Time-PCR.pdf
The so-called SimpleProbe format requires only one hybridization probe, labeled with only one fluorophore (Fluorescein), to achieve sequence specificity. Typically such a probe is designed to specifically hybridize to a target sequence that contains the SNP of interest. Once hybridized to its target sequence, the SimpleProbe probe emits more fluorescence than it does when it is not hybridized. As a result, changes in fluorescence are based solely on the hybridization status of the probe.
In this format you can potentially detect any changes, mutations under the probe. Using SimpleProbes is robust can be used on poor DNA yet requires only one probe per SNP or Indel. It works fantastic on both Indels and SNPs. In my last job, we even succeeded to make 2 different SNP reaction work in the same tube. Still designing and calibrating a SimpleProbe reaction to work can be challenging. And most popular realtime PCR instruments don’t have the required analysis SW. Only Roche have. Also there are very very few oligo vendors that work with the Simpleprobe modification.
Summary
In this technote I mentioned and shared my personal experience with some common Realtime PCR allelic discrimination-genotyping methods. All methods have advantages and disadvantage. Some markers will work easy with Taqman and be a pain with Simpleprobe and others vice versa.
For primers and probes sequence design I recommend always to use a SW tool, there are several out there, including free-online.
Realtime PCR genotyping is best suitable for low to medium throughput. Let’s say hundreds to thousands of samples times several SNPs.
Higher throughput, meaning say hundreds to thousands of samples times tens to hundreds of markers are more suitable on other platforms like Agena or Fluidigm and Douglas Scientific. Screening of 1000’s or even more SNPs are also possible with further platforms like targeted NGS or others but this can be another technote by itself.
