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Results: 3

1.
Figure 1

Figure 1. From: Locked nucleic acid (LNA) single nucleotide polymorphism (SNP) genotype analysis and validation using real-time PCR.

Simulated PCR phase transitions produced from SigmaPlot (version 8.0, SPSS) displaying amplification curves for a true heterozygous genotype. The theoretical behaviour of PCR amplification is determined by the initial ground (IG), exponential growth (EG), linear growth (LG) and plateau (P) phases, respectively. Based on a four-parameter sigmoid model (equation 1), the variables y0, a, x0 and b are obtained to determine ambiguous genotypes based on three criteria.

Matthew P. Johnson, et al. Nucleic Acids Res. 2004;32(6):e55-e55.
2.

Figure 2. From: Locked nucleic acid (LNA) single nucleotide polymorphism (SNP) genotype analysis and validation using real-time PCR.

Simulation curves produced from SigmaPlot (version 8.0, SPSS) examining changes in PCR amplification efficiency (Δb), net fluorescent signal (ΔRx) and the point of inflection—FDM (Δx0). All simulations represent amplification on one fluorophore channel of the Rotor-Gene 3000™. The x-axis represents the amplification cycle number and on the y-axis is the raw fluorescent value (Rx). (A) Changes in amplification efficiency (Δb) with a constant point of inflection (x0 = 25) and a constant net fluorescent signal (ΔRx = 1.0). (B) Changes in net fluorescent signal gains (ΔRx) with a constant point of inflection (x0 = 25) and a constant PCR amplification efficiency (b = 2.0; E = 1.0). (C) Changes in amplification efficiency (Δb) and net fluorescent signal gains (ΔRx) with a constant point of inflection (x0 = 25). (D) Changes in the point of inflection (Δx0) (FDM) with a constant amplification efficiency (b = 2.0; E = 1.0) and constant net fluorescence signal gain (ΔRx = 1.0).

Matthew P. Johnson, et al. Nucleic Acids Res. 2004;32(6):e55-e55.
3.

Figure 3. From: Locked nucleic acid (LNA) single nucleotide polymorphism (SNP) genotype analysis and validation using real-time PCR.

Allelic discrimination curves produced by the Rotor-Gene 3000™ analysis software. The x-axis represents the amplification cycle number and on the y-axis is the raw fluorescent value (Rx). (A) Example of a true heterozygote (TC) with the amplification curves for both fluorophore channels (JOE, FAM) satisfying the three proposed criteria. (B) Example of a true homozygote (CC) with the amplification curve of the JOE channel satisfying Criteria A and B. (C) Example of a true homozygote (TT) with the amplification of the other channel (FAM) satisfying Criteria A and B. (D) Example of an ambiguous heterozygote. Criteria A and B (allowing a 5% error) were not satisfied for the FAM fluorophore channel, and Criterion C was not met. Hence, a true homozygote (CC) is deduced. (E) Example of another (observational) ambiguous heterozygote. All three genotype criteria were not satisfied for both fluorophore channels (allowing a 5% error). Hence, this sample is excluded from genotypic and allelic association analyses.

Matthew P. Johnson, et al. Nucleic Acids Res. 2004;32(6):e55-e55.

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