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PCR for genotyping uses a polymerase chain reaction to read which version of a gene (allele) a DNA sample carries, usually by whether a product appears and how large it is. The controls decide whether a missing band means “not present” or “the reaction failed”, and a standard end-point reaction cannot separate hemizygous from homozygous animals.
Definition and scope
Genotyping by PCR asks which allele or alleles a sample carries at a defined locus. The reaction is designed so that its products differ between alleles, for example a product only when the mutant allele is present, a product only for the wild-type allele, or two products of different sizes. The Mouse Biology Program at UC Davis lists allele-specific PCR among its genotyping services, describing it as using “primers to amplify only the target allele” and as a way of differentiating wild-type, heterozygous and homozygous genotypes.
The same task is searched under many phrasings (pcr and genotyping, genotyping with pcr, genotyping pcr, pcr genotyping, genotyping by pcr). All describe using amplification to assign a genotype. Detecting and quantifying a sequence in real time is a related but different readout; see TaqMan qPCR.
How the readout works in practice
The Jackson Laboratory (JAX) publishes “JAX Genotyping Protocols Explained: Standard PCR and Melt Curve Analysis” (checked 2026-10-29). Each JAX protocol shows the expected band sizes and an example gel run at JAX. The result is read from a gel or, for a melt curve protocol, from a melting profile. For gel readout, see agarose gel electrophoresis basics; the instrument itself is covered in what is a thermal cycler.
What each lane proves
| Element in the run | What it tells you | Source |
|---|---|---|
| Internal positive control (IPC) band | IPC primers amplify a region of a mouse housekeeping gene, so every mouse DNA sample should give an IPC band. JAX recommends using IPC primers when a protocol includes them because they demonstrate that the PCR is working. | JAX |
| Pseudo-heterozygous (PHet) control | A control made by mixing DNA from the available genotypes, used when only homozygous and wild-type DNA is available. | JAX |
| Expected band sizes | Given per protocol, with an example gel. JAX notes the expected results are based on its own protocol and may vary with different PCR reagents and thermocyclers. | JAX |
| All listed primers | For standard PCR and melt curve protocols, typically all listed primers are used so that both wild-type and mutant products are amplified. | JAX |
The reading rule that follows: a sample with no IPC band has not given a usable result, whereas a sample with an IPC band and no mutant band is informative. JAX states that IPC primers are typically used when a wild-type primer set is not possible, as with a transgenic strain.
Types: end-point PCR, allele-specific PCR and melt curve
Three readouts are in common use for genotyping.
- Standard (end-point) PCR. The reaction is run and the products are read by size on a gel. JAX publishes it alongside melt curve protocols.
- Allele-specific PCR. Primers amplify only the target allele. UC Davis’s Mouse Biology Program describes it as fast and cost-effective for differentiating wild-type, heterozygous and homozygous genotypes, and lists it alongside Sanger sequencing and loss-of-allele analysis as separate services.
- Melt curve analysis. JAX describes it as standard PCR with a fluorescent dye and a melting protocol after cycling. Each peak “corresponds to an amplified product with a characteristic melting temperature”. In JAX’s example, one peak at 83 degrees C is the IPC product and a second peak at 86.5 degrees C corresponds to the Cre transgene. It needs a cycler that can measure melting temperature. Peak interpretation is discussed in qPCR melt curve analysis.
Where it matters for labs
When end-point PCR is not enough
JAX states that “you cannot differentiate hemizygous and homozygous animals with standard PCR” for transgenic strains, and that such strains must use a qPCR protocol to tell them apart. A colony plan that needs zygosity for a transgene therefore needs a dosage assay, not a longer gel. Related worked cases are in lox-stop-lox alleles and Cre-lox driver lines; panels that read several targets in one tube are covered in multiplexed PCR.
What a genotyping record should hold
From the elements above, a run record that can be audited later names the primer set, the expected band sizes (or melt peaks) for each genotype, the controls included (IPC, PHet or other), and the cycler and reagents used, since JAX notes that results and melting temperatures may differ with a different setup. Tissue sampling for the DNA source is covered in mouse husbandry.
Common misunderstandings
- “No band means the animal lacks the allele.” Not unless the IPC band is present; otherwise the reaction may not have worked.
- “Hemizygous and homozygous transgenic animals give different gel patterns.” Standard PCR often only determines presence or absence of the transgene, according to JAX.
- “Another lab’s melt temperatures should match ours.” JAX says melting temperatures can vary in a different facility with a different PCR setup.
- “PCR genotyping and sequencing are the same service.” UC Davis lists allele-specific PCR and Sanger sequencing as distinct services.
FAQ
What is the difference between PCR and genotyping?
PCR is the amplification method; genotyping is the goal of assigning which alleles a sample carries. PCR genotyping combines them by designing the reaction so the product pattern differs between alleles.
What does an IPC band show in genotyping PCR?
It shows the reaction worked. JAX describes IPC primers as amplifying a mouse housekeeping gene so that every mouse DNA sample has an IPC band.
Can standard PCR tell hemizygous from homozygous?
No, according to JAX, which says such transgenic strains must use a qPCR protocol.
What is a PHet control?
A pseudo-heterozygous sample made by mixing DNA from the available genotypes, used when no true heterozygous DNA exists (JAX).
Sources
- The Jackson Laboratory, JAX Genotyping Protocols Explained: Standard PCR and Melt Curve Analysis, checked 2026-10-29.
- Mouse Biology Program, UC Davis, Genetic Analysis Services, checked 2026-10-29.
