Notes

1. Use only calibrated pipets, thermal cyclers, or incubators. Do not use heat blocks, because the microtiter plates will warp, producing unreliable results.

2. Sterile disposable aerosol barrier pipet tips are recommended for each addition and transfer to minimize cross-contamination.

3. Use freshly dispensed DNAase-free mineral oil for reaction overlay.

4. When samples present low Net FOZ signals it may be because of the use of insufficient amount of DNA. It is recommended to measure DNA concentration using the PicoGreen method because DNA quantitation by A260/A280 may lead to an overestimation of the amount of DNA in the sample owing to the presence of RNA contamination. If the DNA concentration was lower than recommended, repeat the reaction using a larger amount of DNA.

5. For newly developed assays, low Net FOZ signal may be caused by primary probes that have optimum temperature other than 63°C. To verify this hypothesis, test the Invader assay including the No Target Blank and 0.1 amol (60,000 molecules) of synthetic targets (see Subheading 3.1.5.), at 63 ± 4°C. If the reaction peak is not between 61 and 65°C, use the nearest neighbor calculation (8,9) as a guideline to adjust the probe length so that it is closer to 63°C. For example, if reaction optimum performance peak is at 58°C, determine number of bases to add in order to increase the reaction peak 5°C. Likewise, determine number of bases to remove from probe if the reaction optimum performance peak is higher than 63°C.

Fig. 3. Data generated from Invader assays for analysis of the GSTM1 gene copy number. (A) Scatter plot analysis of Net FAM FOZ and Net RED FOZ signal. (B) Scatter plot analysis of allelic ratios values. (C) Scatter plot analysis of the normalized allelic ratios values. The cluster of diamonds consists of samples containing two copies of the GSTM1 gene. The cluster of squares consists of samples containing one copy of the GSTM1 gene. The cluster of circles represents the samples that are null for the GSTM1 gene (0 copies).

Fig. 3. Data generated from Invader assays for analysis of the GSTM1 gene copy number. (A) Scatter plot analysis of Net FAM FOZ and Net RED FOZ signal. (B) Scatter plot analysis of allelic ratios values. (C) Scatter plot analysis of the normalized allelic ratios values. The cluster of diamonds consists of samples containing two copies of the GSTM1 gene. The cluster of squares consists of samples containing one copy of the GSTM1 gene. The cluster of circles represents the samples that are null for the GSTM1 gene (0 copies).

6. High background signal in the No Target Blank wells can be caused by the use of FRET and/or primary probe oligos not properly purified. High background signal will negatively impact the Net FOZ signal generation.

7. For probes with unusual performance differences at 63°C, allelic ratios may fall into the equivocal ranges. The criteria used for interpreting allelic ratios in these methods are generic guidelines. Individual laboratories may choose to establish their own allelic ratio ranges for any given assay. Although the allelic ratios for heterozygous samples in the Invader SNP assays or samples with two copies in the gene copy number Invader assay should theoretically be close to one, in practice this ratio varies owing to differences in the performance of the specific allele probes. However, for any given Invader assay, the ratio remains relatively constant within the validated conditions for a given sample preparation method.

8. The gain settings will vary depending on the instrument. Gain setting of the fluorescence plate reader should be adjusted to be in the linear dynamic range of the respective reader.

Acknowledgments

We wish to thank Annie Weber for the experimental data, and Peggy Eis,

Laura Heisler, Hon Ip, and Marilyn Olson for critically reading the manuscript.

References

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