Your DNA may not be accurately quantitated
There may be PCR inhibitors present in your sample
Potential PCR inhibitors can originate from the tissue source of the DNA sample, from the purification method, or from the plastics used during sample preparation. Examples of inhibitors originating from the cell include heparin (Holodiny et al., 1991), proteins, and heme (Akane et al., 1994, DeFranchis et al., 1998). Examples of inhibitors originating from DNA preparation are phenol (Katcher and Schwartz, 1994), proteases, detergents (SDS), and salts.
The presence of polymerase inhibitors can decrease PCR efficiency, leading to:
- Trailing clusters
- No amplification, such that some (or all) samples cluster with the No Template Controls
Applied Biosystems examined the effects of hematin on the TaqMan® Genotyping Assays. Three concentrations of hematin (0.25 µM, 0.50 µM, or 1.00 µM) were added to the reaction volumes of each well of the assay. The results are shown here:
PCR Inhibition as a function of hematin concentration
PCR inhibition effects begin at 0.25 µM hematin. Assay performance is severely compromised at 0.50 µM hematin; however, although signal strength is significantly lowered, it is still possible to call genotypes. Assay performance is entirely inhibited at 1.00 µM, at which there is no cleaving of the probes, resulting in no fluorescence.
The DNA purification method used to prepare the DNA can affect the success of PCR (Maaroufi et al., 2004). Choose a method that minimizes degradation and removes inhibitors. One method for assessing DNA purity is to calculate the A260/A280 ratio. In addition, absorbance at 230 nm can indicate the presence of phenol (Gallagher, 1994).
In Applied Biosystems laboratories, A260/A280 ratios between 1.8 and 2.0 indicate that the genomic DNA samples are pure enough for use with TaqMan® Genotyping Assays.
- Dilute the sample and run the assay with the diluted sample. If the inhibition decreases, then it is likely there are PCR inhibitors in the sample.
- Re-purify the sample and run the assay again.
- Choose a method that minimizes degradation and removes inhibitors. One method for assessing DNA purity is to calculate the A260/A280 ratio; achieve a ratio between 1.8 and 2.0.