How do I obtain the required sequence data?

1. Sequence your PCR product
2. Ask your collaborator/vendor for the construct sequence
3. Ask around your lab for the construct file

Will the information I have be sufficient for assay design?

We are unable to design from the following forms of sequence data:

1. An estimated reconstruction of your mutant allele
2. Only the primers you are currently using to genotype
3. The sequence of the wild type allele
4. A picture of your mutant allele sequence

Why does Transnetyx need your sequence data?

Transnetyx does not use traditional gel-based PCR protocols for genotyping, therefore we are unable to use already existing protocols from other labs in our system. We are using a real time PCR platform, designing Taqman® fluorescently labelled probes to detect each allele of interest. Because our reactions amplify a very short segment of DNA (60-80bp ideally), when specific mutant probes are needed, this will require us to know the exact sequence at a junction specific to that allele (e.g. endogenous-insert junction, promoter-DNA junction). Designing a probe in this instance can normally be accomplished by obtaining the sequence in between the traditional PCR protocol being used.

Instances where sequence data will usually be needed:

1. You have crossed multiple strains containing the same selectable marker (e.g. neomycin). We will need to design an assay specific to each mutant allele in order to accurately genotype in this situation.
2. You have a conditional mutation. Being that alleles of conditional mutations are very similar, and due to the hairpin nature of loxP sites, we will need to design a specific assay based on the actual sequence of the mutant alleles in order to accurately identify each allele in the system.
3. You need to distinguish between multiple transgenes containing identical functional sequence. We will use your sequence data to design an assay spanning the promoter-transgene junction in this instance.