Quantify single nucleotide polymorphism (SNP) ratio in pooled DNA based on normalized fluorescence real-time PCR

BMC Genomics

Table 1 An example of four-parameter sigmoidal equation modeling and result of fluorescence normalization

Cycle Numberx^a	D2 sample FAM (Raw Rx)^b	D2 Normalized Rx . (Raw Rx -/Raw Y₀-1)^c
23.58	353.48	0.056045583
24.58	355.38	0.061721963
25.58 ^f	367.68	0.098469051
26.58	380.74	0.13748669
27.58	409.2	0.222512879
28.56	429.79	0.284026907
29.61	465.57	0.390922095
30.56	504.15	0.506182473
31.63	535.18	0.598886713
32.63	576.31	0.72176539
33.63	595.56	0.779276076
34.63	639.21	0.909683425
35.63	669.3	0.99957935
Parameter	Sigmoid function fitted for raw fluorescence^d	Sigmoid function fitted for normalized fluorescence^e
a	525.5884	1.5702
b	3.2674	3.2674
X0	33.4481	33.4481
Y0	334.7204	0
R	0.998805	0.99880514

^a amplification cycle number
^b Raw FAM fluorescence data
^cNormalized fluorescence = Raw Rx/Raw y_o-1
^d 4 parameter sigmoid function fitted for raw fluorescence
^e 4 parameter sigmoid function fitted for normalized fluorescence
^f bold text cycles are selected PCR exponential phase for allele ratio quantification

ISSN: 1471-2164