Step 3. Amplify DNA and collect allelic discrimination data
To ensure high quality, reliable, and reproducible data when SNP genotyping samples isolated from FFPE tissues, it is critical that all reactions contain the same input of functional DNA template. Since FFPE samples are often finite, use the lowest recommended input of 1 ng of DNA per reaction (as determined by RNase P detection). For DNA samples with <5% functional template, do not use more than 20 ng of DNA as measured by A260, because increasing the input beyond this amount will not increase the chance of success. Include at least two no-template controls and known genomic DNA controls on each plate to ensure optimal performance of the SNP genotyping assays. To increase throughput, multiple SNP genotyping assays may be run on one reaction plate.
Sequence-specific forward and reverse primers and probes are then used to amplify and detect the SNP of interest. Optimal fluorescence signal is obtained by amplifying 1 ng of functional DNA template (as determined by RNase P assay) for at least 50 cycles. The resulting allelic discrimination data is collected by performing an endpoint plate read which measures the raw fluorescence signal produced by each reporter dye.
>> Recommendation: Amplify 1 ng of functional DNA template (as determined by RNase P detection) for at least 50 cycles on a 7900HT Fast PCR System or a GeneAmp® PCR System 9700. Then perform a post-PCR plate read on the 7900HT Fast PCR System.
Ten genotyping assays were selected from a preliminary forensic identification panel of 19 small nucleotide polymorphisms (SNPs) having minor allele frequencies of >10% , so that all 3 genotypes would be detected in the tested FFPE sample panel. The amplicon lengths of the selected assays ranged from 65–143 bp.
For each SNP assay, 5 µL reactions were performed in duplicate 384-well plates using 1 ng DNA input based on both UV absorbance (A260) and RNase P assays. Amplification was carried out for 40 or 60 cycles in standard mode on a 7900HT Fast Real-Time PCR System.
Despite quantifying functional DNA using the RNase P assay, late cycle signal detection and a failure to reach the plateau phase were observed in ~50% of samples (Figure 2 below). Both of these observations suggest that 40 amplification cycles were insufficient and increased the risk of undetermined or inaccurate results when using the AutoCall feature of the Sequence Detection Systems (SDS) software. Since the assay results were acquired from an end-point plate read, it was essential that the signal was in plateau phase of amplification. Therefore allelic discrimination plots of samples amplified for 60 cycles were analyzed to investigate if additional cycles could improve fluorescence signal and data quality. Figure 1C demonstrates that tighter genotype clusters with reduced trailing can be obtained by using 1 ng of DNA (as determined by RNase P detection) and amplifying for at least 50 cycles. In fact, some of the DNA samples that clustered near the no template controls because of low signal can now be genotyped (Figure 1C).
Figure 2. Amplification Plots of RNase P Quantified FFPE DNA after 60 Cycles. Using TaqMan® SNP Genotyping Assay, 1 ng by RNase P detection of each FFPE DNA sample (n=100) was amplified for 60 cycles. Real Time PCR data indicates that the signal intensities of more than half the FFPE DNA samples that clustered near the no template controls after 40 cycles reached plateau after at least 50 cycles. The genotype of these samples can now be AutoCalled by the SDS Software.
Figure 4. Achieve Highly Reproducible and Reliable Data from TaqMan® SNP Genotyping Assay. 1 ng, as quantified by RNase P detection, of each human FFPE DNA sample (n=105) was amplified in replicate for 60 cycles followed by AutoCalling using SDS 2.2.1 Software. Gene Symbol: GABRA2, Assay ID: C___8263011_10
1. Kidd K, Andrew J. Pakstis, William C. Speed, Elena L. Grigorenko, Sylvester L.B. Kajuna, Nganyirwa J. Karoma, Selemani Kungulilo, Jong-Jin Kim, Ru-Band Lu Adekunle Odunsi, Friday Okonofua, Josef Parnas, Leslie O. Schulz, Olga V. Zhukova and Judith R (2006) Developing a SNP panel for forensic identification of individuals, Forensic Sci Int 164(1):20–32.