SNP Not Amplifying

• Your DNA may not be accurately quantitated
• You may have degraded genomic DNA
• There may be inhibitors present in your DNA sample
• Your reagents may have been mishandled or may be expired
• You may not have ROX™ dye in your master mix
• There may be an error in your reaction setup

The amount of genomic DNA is critical to the success of the assays. Within an assay and/or study, uniformity of genomic DNA concentration leads to accurate, robust, and reproducible results, and ensures efficient use of valuable samples. Variability in genomic DNA concentrations can lead to experimental anomalies that may affect interpretation of genotyping results, as shown in the following figure. Precise handling and quantitative measurements before running an assay can prevent possible errors without waste of reagents and samples.

Allelic discrimination plots for the TaqMan Genotyping Assay C_1204092_20

Solution:

• Always perform concentration measurements before using any genomic DNA, even commercially prepared DNA.
• Use the recommended amount of genomic DNA (3 to 20 ng) per sample per assay.
• Always use the same quantity of genomic DNA for all samples of an assay on a plate.
  Commercially purchased DNA comes with concentration information, but it is good practice to confirm DNA concentrations in your laboratory. Applied Biosystems scientists have found that the concentrations of DNA listed for commercially available genomic DNA can be quite different from laboratory measurements.

There are numerous methods for quantitating genomic DNA, including:

• UV spectroscopy
• Absolute quantitation using a known copy number gene such as TaqMan RNase P
• Fluorometric analysis

Applied Biosystems recommends UV spectroscopy or the TaqMan RNase P method for DNA quantitation.

UV Spectroscopy

UV Spectroscopy is the most widely used method for quantifying DNA of all types. Even though it is most widely used, the consumable reagents used in the process vary greatly.

• Ensure that the spectrophotometer is set up correctly for the reagents to be used.
• Background constants for optical plastic cuvettes and plates are different from those of quartz cuvettes and plates; consult the instrument manual for ways to determine background constant.
• Be cautious of the diluents used with the genomic DNA samples. They can have differing properties which may affect the final results.
• Use UV spectroscopy can to quantitate genomic DNA by reading sample absorbance at 260 nm (A₂₆₀). The A₂₆₀ is most accurate when your nucleic acid is pure, and is most useful for DNA in microgram quantities (Gallagher 1994)
• Take a concurrent reading at 280 nm to determine the concentration of contaminating proteins; the A₂₆₀/A₂₈₀ ratio is used to determine purity of a DNA sample. Proteins, particles in the solution, and aromatic chemicals can affect the reading.

The effective read range of UV spectroscopy is 0.1 to 0.999 which corresponds to approximately 4 ng/µL to 50 ng/µL of genomic DNA. Values above or below that range are invalid absorbance readings.

• To ensure accurate quantitative results, dilute your genomic DNA samples so that the A260 reading is between 0.1 and 0.999 (remember to record the dilution factor and the diluents used).
• Ensure that there is enough genomic DNA to use this method; most plates and cuvettes have minimum working volumes, and the genomic DNA sample used for the quantitative measurement will be discarded.     

Absolute Quantitation

Absolute quantitation measures the total amount of amplifiable genomic DNA. This technique requires the creation of a standard curve using genomic DNA samples of known concentrations. The standard samples must be pre-quantitated and validated using an independent method such as spectrophotometry or fluorometry. The unknown samples are compared to the known samples for quantitation.

Two well-known techniques for absolute quantitation are:

• TaqMan Assay Chemistry
• SYBR Green Assay

Absolute quantitation using the TaqMan technology is a highly accurate technique for quantifying DNA. The TaqMan DNA Template Reagents and the TaqMan RNase P Detection Reagents provide convenient methods to quantitate genomic DNA. The kits include pre-diluted and validated standards at five concentrations per kit: 0.6 ng/µL, 1.2 ng/µL, 3.0 ng/µL, 6.0 ng/µL, 12.0 ng/µL. Dilute or aliquot to the appropriate range for the samples.

SYBR Green is a dye that is a minor groove binder which binds only to double-stranded DNA (dsDNA). This method is less specific than the TaqMan method because the dye will bind to any dsDNA. The use of this method will require melt curve analysis to verify the specificity of the assay.

For either technique, be sure to run the standard curve and unknown samples on the same plates in the SDS instrument.

Fluorometric Analysis

Quantitation of DNA by fluorometric analysis uses various intercalating dyes. These are summarized in the table below:

 Dye features

Degradation can result from:

• Using very old DNA samples
• Using DNA extracted from formalin-fixed paraffin embedded samples
• Freezing and thawing DNA samples repeatedly
• Leaving DNA samples at room temperature
• Exposing DNA samples to heat or physical shearing
• Purifying DNA samples inefficiently so residual nucleases remain

Solution:

Run an agarose gel to determine if the DNA is degraded. Look for a tight band of high molecular weight; smearing indicates degraded DNA.

Agarose gel stained with ethidium bromide showing heat degradation of genomic DNA. This gel shows progressive degradation with increasing time of two human genomic DNA samples subjected to heating at 99°C for 0 to 30 minutes.

As the average size of the DNA in a degraded sample approaches the size of the target sequence, the amount of PCR product generated is reduced. This is due to the reduced number of intact templates in the size range necessary for amplification. An example of assay results using DNA degraded by heating is illustrated below. Genomic DNA degraded by other causes will also deliver poor assay results.

Allelic discrimination clustering on a degraded sample 

Factors that affect DNA degradation include tissue preservation methods, exposure to UV radiation, temperature, pH, and salt concentration of the environment (Dean, M. and Ballard, J.W.O., 2001). There are many sources of genomic DNA including fresh capillary blood, buccal scrapes, solid organ biopsies, and paraffin-embedded tissue. The table below provides recommended sample storage conditions to help minimize DNA degradation.

Recommended sample storage conditions

Use more caution in interpreting the results if the DNA is substantially degraded. If possible, consider repeating the assay using freshly prepared genomic DNA samples.

Potential PCR inhibitors can originate from the tissue source of the DNA sample, the purification method, or 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 scientists examined the effects of hematin on the TaqMan Genotyping Assays. Hematin (0.25 µM, 0.50 µM, or 1.00 µM) was added to the reactions in each well. The results are shown below:

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 hematin, 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.

Solution:

• 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 of between 1.8 and 2.0.

The use of mishandled or expired reagents may result in:

• Some or all samples clustering with the NTCs
• Trailing clusters
• Weak overall reaction (weak signals)

Solution:

Perform the assay again with newly prepared reagents, following the handling guidance below:

Assay Considerations

It is important to:

• Store TaqMan SNP Genotyping Assays at –15 to –25 °C when they are not in use.
• Minimize freeze-thaw cycles to no more than five cycles. Too many freeze thaw cycles can cause cleavage of the dye from the probe.
• Limit the assay exposure to light. The fluorescent dyes are susceptible to photo bleaching. Photo bleaching can result in a lower overall signal for the reaction.

TaqMan Genotyping and Universal PCR Master Mix Considerations

• Store TaqMan PCR Master Mixes at 2–8 °C.
• Prior to use, make sure the Master Mix is thoroughly mixed.

The use of a PCR Master Mix that does not contain ROX™ dye (or a similar passive reference) can cause:

• Trailing clusters
• Some or all data is undetermined (there is an "X" instead of a called allele in the AD plot).
• Diffuse clusters

Solution:

• ROX™ dye is a passive reference dye that improves the precision of the results by compensating for small fluorescent fluctuations, such as bubbles and small well-to-well variations.

Note: The Sequence Detection Software on the 7900HT Fast Real-Time PCR System will not call the alleles when ROX™ dye (or another passive reference) is not present.

• Use Applied Biosystems TaqMan Genotyping Master Mix or TaqMan Universal PCR Master Mix, both of which contain ROXÔ passive reference.
• Confirm ROXÔ passive reference is specified in SDS software:

Specify ROX™ passive reference in SDS software

DNA or assay missing from reaction well

If genomic DNA or one of the assay reagents is not added to the reaction well, no PCR amplification takes place and the sample clusters with the NTCs.

Solution:

Perform the assay again, making sure to:

1. Pipette carefully
2. Mix thoroughly

Evaporation of the reaction

Evaporation of the reaction can occur if the reaction plates are not properly sealed, leading to:

• Outliers (mild/moderate evaporation)
• Trailing clusters (moderate evaporation)
• Samples clustering at the NTC (extreme evaporation)

Evaporation can occur if the plate is not properly sealed. The loss of water during evaporation leads to an increased concentration of the dyes. This results in an increase in the signals from the reporter and ROX™ dyes. The degree of evaporation influences the assay results:

• Mild:  If the PCR reaction is not affected, the ROX™ dye can compensate for the increased signals and the assay will work correctly
• Mild to moderate:  Outlier samples may be observed. Depending on the number of wells affected, the plot may show only a few outliers or it may show a trailing cluster.
• Extreme evaporation occurring early in the reaction:  The PCR reaction fails and the samples cluster with the NTC.

Solution:

1. Check the location of the wells for the problem calls. Evaporation can most often occur around the edges of the plate.
2. Check the seals of the optical adhesive cover for leaks.
3. If there are leaks, use a MicroAmp Adhesive Film Applicator to thoroughly seal the cover. Make sure to run the applicator over the edges of the seal.
4. Perform the assay again