Small Amplicon HRMA employs two primers closely surrounding the SNP of interest (8). Amplicon size is kept at a minimum, between 50–70 bp.
The hydrogen bonding characteristics of the nucleotide at the SNP position alters the melting temperature of the small amplicon, which can be detected by HRMA. The melting temperature differentials of homozygous Small Amplicons are reported to vary from 0.8 – 1.2 C, while heteroduplex amplicons are reported to melt at 2.0-3.0 C lower temperatures, as shown in Supplemental Figure S-1B.
Blocked (Unlabeled) probe HRMA employs a 3′phosporylated oligonucleotide probe that overlaps the SNP of interest but cannot be extended in the PCR reaction, and two outer primers that produce a 65 – 150 bp amplicon (9). By including a limiting amount of primer on the same strand as the probe, asymmetric PCR produces an excess of opposite strand product to serve as a probe binding partner during HRMA. Probes form either perfect match or mismatch duplexes, with reported melting temperature differentials of 5.0 – 8.0 C, as shown in Supplemental Figure S-1C.
Small amplicon primer sets for 11 SNPs, and blocked probe primers and probes for 33 SNPs were developed using online Primer3 design software (http://frodo.wi.mit.edu/primer3/). Small amplicon and blocked probe primer sets are available in Supplemental Tables S-2 and S-3, respectively. PCR and HRMA were performed in 96-well plates using Platinum Taq and 1x LCGreen Plus+ DNA intercalating dye (Idaho Technologies, Salt Lake City, UT, USA) on a LightCycler 480 platform (Roche Applied Science, Indianapolis, IN, USA). HRMA results were evaluated using LightCycler 480 Software Version 1.5.
LC480 Cycling Parameters were as follows: Initial Denaturation: 95°C (8 min); Amplification: 60 cycles of 95°C denature (4 s), 65°C annealing (6 s), 72°C extension (12 s); Melt: 95°C denature (15 s), 50°C anneal (60 s), 50–95°C continuous ramp at 0.06°C/second, 10 measurements per degree. Cooling: 50°C (10 s).
Each primer set was evaluated with B6, C3H, and 1:1 mixed B6 + C3H (obligate heterozygous) genomic DNA, as well as an H2O negative control.
Due to different assay design requirements, individual SNPs were not generally compatible with all three methodologies. SNPs were selected from the Perlegen2 database (10). The Sanger Mouse Genomes Project database provides a useful additional resource to select or verify candidate SNPs for genotyping (6).Results and discussion
The generation of congenic mouse strains is an iterative process. The ability to detect unique meiotic recombination events producing a novel isolated genomic interval in individual mice in a timely and cost effective manner is essential for efficient progress. By definition, an average of 25 out of 100 pups are expected to contain a unique crossover event within a 25 cM locus. Therefore, as targeted intervals decrease in size, the number of expected new recombinants also decreases, leading to diminishing returns and increasing colony management costs. Theoretical calculations indicate that to narrow a single 25 cM locus to 5 cM by breeding of ISCLs requires approximately 300 individuals, while further reducing each locus from 5 cM to 1 cM requires approximately 380 additional individuals per regulatory gene (11). For the complex Bbaa2 locus, with multiple putative regulators within a 20+ cM interval, this predicts that approximately 1820 individuals, the vast majority of which will be non-informative, will be required to achieve our goal of 1 cM resolution for each putative locus.
Congenic breeders and their young pups can be maintained in a single cage, but are generally separated into 3 or more cages at weaning age. Since so many pups are non-informative as congenic intervals become narrow, rapid genotyping that allows efficient culling prior to weaning age may therefore provide a multiplier effect on cage cost savings, depending on the unique characteristics of individual colonies and institutional protocols. This process greatly benefits from the flexibility afforded by a high-throughput genotyping assay that can be rapidly and inexpensively performed on either small or large numbers of samples and can be targeted specifically to regions of interest within a sub-interval.
We previously relied on a panel of 11 microsatellite markers, some of which were poorly spaced or tightly clustered together, to genotype Bbaa2 recombinants. To improve upon this, several methods of SNP genotyping were evaluated.