Journal: Human Gene Therapy
Article Title: Allele-Specific Quantitative PCR for Accurate, Rapid, and Cost-Effective Genotyping
Figure Lengend Snippet: Validation of ASQ genotyping of the nr3c1 locus with restriction fragment length polymorphism (RFLP). (A) nr3c1 allele discrimination plot was generated by CFX Manager software. A total of 40 samples were used for this validation. Clustering of alleles visually assists allele scoring. Two no-template controls (NTCs) were used that clustered near the origin of the graph. Six samples of a serial dilution of wild-type (WT) genomic DNA were used for quality control (3×; 1-, 3-, 9-, 27-, 81-, and 243-fold dilution). The samples with the lowest concentrations (81- and 243-fold dilution) did not produce a valid signal, resulting in two other unscored data points (black diamond) on the graph. *The sample 25 evaporated during ASQ reaction and was discarded from ASQ genotype scoring. The original allele calls and standard curve are available in Supplementary Fig. S4 . (B) nr3c1 allele scoring with RFLP. The total amplicon is 685 bp with two restriction sites for the restriction endonuclease, PvuII -HF. A homozygous allele gives 383 and 155 bp bands, a heterozygous allele 383, 241, 155, and 142 bp, and a WT allele 241, 155, and 142 bp. L, molecular ladder; x, empty well. (C) qPCR amplification plot. FAM plot (blue) shows amplifications of a WT allele, whereas HEX plot (dark green) shows amplifications of a mutant allele.
Article Snippet: Taking advantage of generic software provided with a qPCR machine, we used an allele discrimination plot automatically constructed by CFX Manager.
Techniques: Generated, Software, Serial Dilution, Amplification, Real-time Polymerase Chain Reaction, Mutagenesis