Preparing New Targets
Expanding on the Ca2LF Concept to Develop SNflaPs
In the project Ca2LF, we developed the Flappase assay to detect a single nucleotide polymorphism (SNP) in one gene, the beta-casein gene. The detection of the SNP would only tell us what version of the beta-casein allele is present, A1 or A2. For this year’s project we wanted to expand the usefulness of our genetic testing system. We decided to expand our system to be used to characterize the genetic profile of a cow on a variety of different traits so that farmers could make informed decisions regarding a variety of advantageous and deleterious traits when breeding their cows.
Identifying potential target genes
We began by interviewing two experts in cattle genetics and breeding, Dr.’s Dechow and Huson. During this interview, we learned about some genetic conditions that are not desirable in cattle, such as diseases, conditions that negatively impact fertility, and physical traits (for example, being horned) that farms routinely attempt to breed out of their herds. We also learned about genetic traits that are advantageous and routinely selected for by farmers when breeding, including genes involved in promoting fertility, milk production, and milk composition. We compiled a list of these genes and researched their different alleles (Table 1).
TABLE 1 HERE
Another important piece of feedback we received from the interviews with Drs Dechow and Huson is that most alleles are created by multiple SNPs, or larger changes to the gene, such as insertions, deletions, inversions, or duplications. So, to be useful, our SNflaPs genetic testing system needs to detect multiple types of polymorphisms. We decided to focus our efforts on selecting a few genes, each of which has a different type of polymorphism, to serve as proof of concept that the Flappase-based detection system can discriminate between these alleles.
Table 2. Genes selected for proof-of-concept testing of the Flappase assay for detecting polymorphisms. The genes selected for each contain different polymorphisms, or multiple SNPs found in close, medium, or far proximity to each other.
TABLE 2 HERE
When deciding which genes to use for testing the detection system we used the following criteria:
- The length of any polymorphism should be less than 5,000 bp.
- If multiple SNPs are to be detected, the distance between these should be enough to fit both the oligos, as well as accommodate the Flappase protein that is approx. 22 nucleotides and 75 Angstroms apart (See modeling page).
- For initial testing, any gene selected should be of sufficient length to be synthesized for the creation of positive control DNAs.
Based on these characteristics, we decided to begin by pursuing LRP4 and APOB as model genes.
Designing and cloning new targets and oligos for use with the Flappase system
New Target #1- LRP4
The LRP4 gene encodes for LDL receptor related protein 4 (3). Two well-characterized allelic variants of this gene have been identified, the normal (wildtype) version, and a syndactylous version. Inheritance of the syndactylous version leads to congenital syndactyly. This condition, also known as mulefoot, refers to the fusion or non-division of the two developed digits of the bovine foot (4).
Figure 3: Sequence variation between the normal and syndactyl alleles of LRP4. The two versions differ by two SNPs, where nucleotides CG are substituted by AT at positions 4863 and 4864.