Genotyping
Allele-specific PCR: two reactions per sample, each with a primer whose 3′-terminal base sits on one allele, so the genotype is read from which reaction produced a product, with an internal control amplicon in both to show the reaction worked.
Our ruleSimulations prepare you for the bench but don't replace hands-on practice.
PCR genotyping
Allele-specific PCR: two reactions per sample, each with a primer whose 3′-terminal base sits on one allele, so the genotype is read from which reaction produced a product, with an internal control amplicon in both to show the reaction worked.
How it differs from the standard method
- The variant base must be the primer's 3′-terminal base: both the duplex destabilisation and the polymerase's reluctance to extend a mismatched 3′ end then work for you.
- Discrimination is a rate difference, so it degrades as the annealing temperature falls and as cycles accumulate. Optimise Ta against known genotypes and stop at 30–35 cycles.
- An internal control amplicon in every reaction is not optional: without it, no band means either 'allele absent' or 'reaction failed'.
- Titrate the two allele-specific primer mixes against a known heterozygote, or one allele will amplify better and heterozygotes will be called homozygous.
- Tetra-primer ARMS puts both allele-specific primers and a flanking pair in one tube, with amplicons of different lengths; it halves the reactions but is much harder to balance.
1A genotype is a call, not a measurement
Genotyping asks a closed question: at a position you already know about, which of the possible alleles does this sample carry? Because a diploid sample carries two copies of the position, there are three answers (homozygous for one allele, homozygous for the other, or heterozygous) and the whole design problem is building an assay that returns three distinguishable outcomes rather than two.
2Where discrimination comes from in an allele-specific primer
A primer whose 3′-terminal base sits on the variant discriminates for two reasons at once. The mismatch destabilises the duplex, so at a given annealing temperature the primer spends less time bound to the wrong allele; and, more importantly, the polymerase has to extend from that 3′ end, and a mismatched 3′ terminus is a poor substrate.
Answered by a reaction working or not
Two reactions per sample. Needs an internal control amplicon in each, or an empty lane is ambiguous between 'allele absent' and 'reaction failed'.
Answered by fragment sizes
One amplicon for everyone, then an enzyme whose site the variant creates or destroys. Needs the digest to go to completion.
Answered by a ratio of two colours
Two labelled probes in one well. Heterozygotes cleave both and land between the two homozygote clusters.
3Reading a cluster plot
In a two-probe endpoint assay each well gives two numbers: how much of each dye was released. Plot one against the other for the whole plate and the samples fall into three groups.
4Allele dropout, and why a confident homozygote is the dangerous answer
If the two alleles do not reach the readout in similar amounts, a heterozygote is reported as a homozygote. There are two routes to that.
- Everyone is a heterozygote
- The discrimination failed, not the biology. Check the primer design (is the variant at the 3′ end?), the annealing temperature and the cycle count; in RFLP, check whether the digest went to completion, because residual uncut product adds the 'other allele' band to every lane.
- Everyone is homozygous for the variant (RFLP)
- Nothing was cut. The enzyme is dead, the buffer is wrong, or there is no site in the amplicon at all. The known homozygote control for the cut allele is what catches it.
- A heterozygote with one faint band
- Preferential amplification. Titrate the two allele-specific primer mixes against a known heterozygote; if one allele is consistently weaker, redesign rather than compensating.
- No-calls scattered across the plate
- Usually variable template quality rather than a design problem. Quantify and normalise the input; a no-call rate above about 5% means the assay needs work, not that the samples need repeating.
- A band in the no-template control
- Amplicon carry-over. A finished reaction holds around 10¹² copies, so aerosols from the post-PCR bench easily reach a master mix. Nothing on the plate is reportable until a blank is clean.
Key terms
Practise these as flashcards →One of the alternative sequences found at a given position. A diploid sample carries two, which may be the same (homozygous) or different (heterozygous).
Amplification refractory mutation system: a primer whose 3′-terminal base sits on the variant, so it extends efficiently on one allele and poorly on the other.
Restriction fragment length polymorphism: a variant that creates or destroys a restriction site, read as a change in fragment sizes after digesting a PCR product.
The two-dimensional scatter of endpoint fluorescence in the two probe channels, in which genotypes appear as clusters rather than as values.
Failure of one allele to reach the readout, from sampling too few molecules or from preferential amplification, which reports a heterozygote as a homozygote.
The honest outcome when the data do not place a sample in a genotype. A no-call is a result; a miscall is not.
A second product in the same reaction, from primers that ignore the variant, so a missing allele-specific band can be distinguished from a failed reaction.
In array and probe genotyping, the normalised ratio of the two allele signals; clusters are defined in this coordinate rather than in raw intensity.
The fraction of attempted genotypes that produced a call. Together with concordance on duplicate samples, it is the standard quality metric for a genotyping run.
Comparing the observed genotype counts with those expected from the allele frequency; a strong departure usually means a technical problem such as a lost cluster, not population structure.
When to use it
- Confirming the genotype of a mouse or plant line at a known engineered or natural locus, where the alleles are defined in advance.
- Screening a cohort for a handful of known variants, where sequencing every sample would be far more expensive than a targeted assay.
- Checking a clinically or agronomically important variant in a defined assay, where a validated, auditable call matters more than discovering anything new.
- Genome-wide association or ancestry work, where an array measures hundreds of thousands of known positions per sample.
- Verifying a cell line's identity by typing a panel of known polymorphic markers.
Compared with alternatives
Reads the actual sequence, so it finds variants you did not know about and resolves ambiguous genotypes; far more expensive and slower per sample, so it is the confirmation method rather than the screen.
The amplification step underneath most genotyping formats. Genotyping adds whatever makes one allele distinguishable from the other.
The cutting step RFLP depends on, and the source of its main failure mode: a digest that has not gone to completion imitates heterozygosity.
The same instrument and probe chemistry used to measure how much rather than which: a genotyping assay reads the endpoint and ignores the Cq entirely.
Partitioning counts molecules, so it measures allele fractions well below 50%: the method for somatic variants and mosaicism, where a genotype call makes no sense.
Gives every position including structural variants; genotyping arrays and assays remain much cheaper per sample for known positions and for very large cohorts.
The practical application in a colony: ear or tail sampling, crude lysates, and a need for a robust assay rather than a sensitive one.
Sub-techniques
Choosing one here switches the whole page, including the protocol and the lab, to it.
Allele-specific PCR: two reactions per sample, each with a primer whose 3′-terminal base sits on one allele, so the genotype is read from which reaction produced a product, with an internal control amplicon in both to show the reaction worked.
- The variant base must be the primer's 3′-terminal base: both the duplex destabilisation and the polymerase's reluctance to extend a mismatched 3′ end then work for you.
- Discrimination is a rate difference, so it degrades as the annealing temperature falls and as cycles accumulate. Optimise Ta against known genotypes and stop at 30–35 cycles.
- An internal control amplicon in every reaction is not optional: without it, no band means either 'allele absent' or 'reaction failed'.
- Titrate the two allele-specific primer mixes against a known heterozygote, or one allele will amplify better and heterozygotes will be called homozygous.
- Tetra-primer ARMS puts both allele-specific primers and a flanking pair in one tube, with amplicons of different lengths; it halves the reactions but is much harder to balance.