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Rapid and Efficient Zebrafish Genotyping Using PCR with High-resolution Melt Analysis
Published on: February 5, 2014
Genotyping common FSHR polymorphisms based on competitive amplification of differentially melting amplicons (CADMA)
Tanni Borgbo1, Lasse Sommer Kristensen, Ida Lindgren
1Department of Biomedicine, University of Aarhus, Aarhus, Denmark, tanni.borgbo@gmail.com.
Purpose:
To provide an improved platform for simple, reliable, and cost-effective genotyping.
Background:
Modern fertility treatments are becoming increasingly individualized in an attempt to optimise the follicular response and reproductive outcome, following controlled ovarian stimulation. As the field of pharmacogenetics evolve, genetic biomarkers such as polymorphisms of the follicle stimulating hormone receptor (FSHR) may be included as a predictive tool for individualized fertility treatment. However, the currently available genotyping methods are expensive, time-consuming or have a limited analytical sensitivity. Here, we present a novel version of "competitive amplification of differentially melting amplicons" (CADMA), providing an improved platform for simple, reliable, and cost-effective genotyping.
Methods:
Two CADMA based assays were designed for the two common polymorphisms of the FSHR gene: rs6165 (c.919A > G, p. Thr307Ala, FSHR 307) and rs6166 (c.2039A > G, p. Asn680Ser, FSHR 680). To evaluate the reliability of the new CADMA-based assays, the genotyping results were compared with two conventional PCR based genotyping methods; allele-specific PCR (AS-PCR) and Sanger sequencing.
Results:
The genotype frequencies for both polymorphisms were 35 % (TT), 42 % (CT), and 23 % (CC), respectively. A 100 % accordance was observed between the CADMA-based genotyping results and sequencing results, whereas 5 discrepancies were observed between the AS-PCR results and the CADMA-based genotyping results. Comparing the CADMA-based assays to (AS-PCR) and Sanger sequencing, the CADMA based assays showed an improved analytical sensitivity and a wider applicability.
Conclusions:
The new assays provide a reliable, fast and user-friendly genotyping method facilitating a wider implication in clinical practise.
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