Animal genotyping
Genotyping from a small biopsy: an ear punch (preferred, as it also identifies the animal) or a tail tip of no more than about 2 mm in young pups, lysed and used directly as PCR template. Older animals and larger biopsies need anaesthesia and analgesia under the approved protocol.
Our ruleSimulations prepare you for the bench but don't replace hands-on practice.
Tail / ear biopsy PCR
Genotyping from a small biopsy: an ear punch (preferred, as it also identifies the animal) or a tail tip of no more than about 2 mm in young pups, lysed and used directly as PCR template. Older animals and larger biopsies need anaesthesia and analgesia under the approved protocol.
How it differs from the standard method
- Ear punch doubles as identification.
- Tail tip only in young pups, with the length kept to a minimum.
- Crude lysate, 1–2 µL per 20 µL reaction.
1Knowing which animal carries which allele
A transgenic or knockout colony is only useful if every animal's genotype is known: it decides which animals breed, which go into an experiment and which group they belong to. A small piece of tissue, usually the ear punch that also marks the animal's identity, is digested into a crude lysate, and a PCR designed around the modified allele reports what is there.
Wild type
Only the wild-type product: both copies of the gene are intact.
Heterozygote
Both products at similar strength: one copy of each allele.
Knockout
Only the cassette product: both copies carry the modification.
2Why a crude lysate is enough, and how it fails
PCR needs only nanograms of DNA, and an ear punch digested in hot alkali gives far more than that. The cost is that the lysate also carries salts, hair, collagen fragments and blood components that inhibit the polymerase, so adding more lysate can give less product.
3Carry-over, controls and the call
PCR amplifies a few percent of foreign DNA to a visible band, so a punch carried from one animal to the next without cleaning turns wild types and knockouts into apparent heterozygotes, usually with one band much fainter than the other. Known wild-type, heterozygous and knockout DNA on every gel show that both products form and separate, and a no-template control shows the reagents are clean.
4The 3Rs in this procedure
Genotype only what you must
Where a line can be maintained as homozygous or tracked with a visible marker, routine genotyping of every pup may not be needed; the experiment's question, not habit, decides.
Breed and sample to need
Plan breeding from the expected Mendelian ratios so fewer surplus animals are produced, and re-use one sample for every assay rather than taking a second.
The least invasive sample
An ear punch that also serves as the animal's identification is preferred over a tail snip; any tail biopsy is kept small and done at the youngest permitted age, with anaesthesia and analgesia where your licence requires it.
Key terms
Practise these as flashcards →A 1–2 mm hole punched in the ear; the pattern of holes identifies the animal and the tissue goes to genotyping.
Digesting tissue in hot sodium hydroxide, then neutralising with Tris, to release PCR-ready DNA in under an hour.
One common primer with an allele-specific primer for each allele, giving products of different sizes in one tube.
Anything in the sample that blocks the polymerase: salt, heme, collagen, residual alkali or active proteinase K.
DNA transferred from one sample to the next on instruments or gloves, which PCR then amplifies.
The 1 : 2 : 1 wild type : heterozygote : knockout proportion expected from a heterozygous cross.
When to use it
- Assigning genotypes to pups at weaning so that breeding and experimental groups are correct.
- Checking for an unexpected ratio of genotypes, which can reveal embryonic or perinatal lethality.
- Confirming the genotype of experimental animals again at the end of a study.
Compared with alternatives
For single-nucleotide variants, allele-specific PCR, RFLP or TaqMan assays read a base change rather than an insertion.
The underlying amplification; the PCR guide covers primer design, cycling and troubleshooting in depth.
How the products are separated and sized, including how agarose concentration sets resolution.
Sub-techniques
Choosing one here switches the whole page, including the protocol and the lab, to it.
Genotyping from a small biopsy: an ear punch (preferred, as it also identifies the animal) or a tail tip of no more than about 2 mm in young pups, lysed and used directly as PCR template. Older animals and larger biopsies need anaesthesia and analgesia under the approved protocol.
- Ear punch doubles as identification.
- Tail tip only in young pups, with the length kept to a minimum.
- Crude lysate, 1–2 µL per 20 µL reaction.