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Sanger DNA Sequencing: Template Preparation

 

Template purity and concentration are the two most important factors in obtaining optimal results with automated fluorescent capillary sequencing. Low-quality template DNA or a suboptimal quantity of template DNA will have a significant impact on the success of the sequencing reaction.

If you are preparing DNA templates (plasmid DNA, PCR amplicons) for Sanger DNA Sequencing, please consider taking advantage of our subject-matter expertise by following our recommendations and guidelines. We would like your Sanger DNA Sequencing samples to work the first time and result in high-quality data - without the necessity to repeat or to troubleshoot!

Preparation of Plasmid DNA Template for Capillary Sequencing:

Today, numerous methods and plasmid preparation kits are available which yield template DNA of excellent quality. Most if not all of these options employ a modified alkaline lysis procedure. A variety of factors, often caused by some common mistakes or omission of specific protocol steps, can compromise the quality of a plasmid DNA template.

Examples include:

• - Contaminants such as salts, organic chemicals (phenol, chloroform or ethanol) and residual detergents
• - Presence of cellular components such as RNA, proteins, polysaccharides or chromosomal DNA
• - Carry-over of silica fines
- Degradation during storage
• - Heterogeneous population of plasmid DNA molecules (contamination with another plasmid)

Low-quality template DNA can result in noisy data (i.e. small secondary peaks underneath the primary peaks in a sequence read), unusable sequence data (i.e. sequence data contains mostly N's), or weak signal. In addition, template DNA of poor quality can reduce the lifetime of the capillary array.

While we are not endorsing any commercially available products, we suggest following plasmid preparation protocols carefully and taking into account the following recommendations:

• Isopropanol-precipitated Plasmid DNA must be washed (at least once as directed) with 70% ethanol to remove excess salt. Residual salt in the final template preparation will have a negative impact on the activity of Taq polymerase.
• The template DNA must be dried completely before its final resuspension in nuclease-free water, in 10 mM Tris-HCl, pH 8.0 (EB Buffer), or in Low TE Buffer (10 mM Tris-HCl, 0.1 mM Na2EDTA, pH 7.5-8.0). Residual ethanol is detrimental to Taq cycle sequencing. If the plasmid DNA purification method involves the use of a spin column, care must be taken to remove any ethanol that might be trapped in the column after the last wash step (prior to eluting the plasmid DNA from the column). We recommend first rotating the column (sitting on the receptacle tube) by 180 degrees and proceeding with a second ("dry") centrifugation step.
• We recommend an additional centrifugation step following spin column elution to remove any residual column resin, which would result in poor sequencing results. After centrifugation of the eluted plasmid DNA for 5 minutes at maximum speed, the uppermost portion of the sample should be transferred into a fresh tube.
• Method-specific directions for cell growth have to be followed to avoid overloading of columns, which would result in low DNA yields and carry-over of undesired contaminants.
  The E. coli host strain used to propagate a plasmid can have a substantial impact on the quality of the purified DNA. Most commonly used strains, such as DH1, DH5α and XL1-Blue consistently produce high quality plasmid DNA. Host strains such as HB101 and its derivatives (such as TG1 and the JM100 series) are known to be a source of variability with respect to template quality.
• We would like to emphasize the importance of following good microbiological practices:
  • You should have a unique DNA product in your sample: Pure "single" bacterial clones should be obtained prior to inoculation of the growth medium (by streaking colonies from transformation plates).
  • The propagation of a plasmid-harboring clone should always be performed in the presence of the appropriate antibiotic.
• We also recommend ensuring the quality of your DNA sample by running a small aliquot on an Agarose gel. There should be one, clearly defined band on the gel representing a particular template. Please make sure to use appropriate gel percentage, separation time and voltage.

DNA Quantitation

As with DNA quality, the quantity of template DNA is an equally important determinant for the success of a sequencing reaction - i.e. it is a prerequisite for obtaining highly accurate and reliable DNA sequence data. Too little template results in reactions with little or no signal and poor or no base calling. Too much DNA causes premature termination of the sequencing reaction, often yielding less than 250 bases of reliable sequence data. Therefore, the accurate quantitation of high-quality template DNA is an important step in the overall sequencing process.

Please take a moment to review our recommendations for determining DNA concentration:

• The DNA concentration should be determined by spectrophotometry when possible.
• Please keep in mind that most spectrophotometers are not at all accurate at OD (Optical Density) values below 0.05. The OD260 values should be in the range of 0.05 to 0.8 to give reproducible and reliable results. Note: Many spectrophotometers can read at 0.05, but only if they are very carefully calibrated with a reference (blank). A plasmid DNA mini-preparation (or PCR; see below) usually does not yield enough DNA to get quantitated accurately on the typical spectrophotometers found in most laboratories. You should be very cautious with your results - unless you can perform the OD measurement with a cuvette volume of 10 ul (or perhaps as high as 100 ul, depending on the specifics of your sample).
• If you are using a NanoDrop UV spectrophotometer, make sure you clean the pedestal frequently and periodically recalibrate the device. If you should observe an unusually high OD when measuring the concentration of a plasmid DNA mini-preparation, you might have RNA or chromosomal DNA in your sample. This is the number one cause of failed sequencing with plasmid DNA mini-preps.
• The 260/280 ratio of your sample should ideally be between 1.8 and 2.0. Values lower than 1.6 and higher than 2.0 indicate contaminants in the sample. These may interfere with the determination of the concentration and might inhibit the sequencing reaction.
• If possible, we recommend measuring the ODs at 230 nm and 320 nm. Both values should be ideally 0.0, and the 230/260 ratio should be lower than 0.6. A high value at OD320 indicates a contaminant.
• If you should not have access to a spectrophotometer or micro-spectrophotometer (NanoDrop), we highly recommend that you run at least two different amounts of your template DNA on an analytical Agarose gel, adjacent to a dilution series (50-500 ng) of a reference DNA fragment of similar size and known concentration, to estimate the concentration of your sample.

DNA Dilution

After determining the concentration, please dilute the template to the correct final concentration (see here) using sterile nuclease-free water or 10 mM Tris-HCl, pH 7.5-8.0. Please do not use any buffers containing Na₂EDTA or divalent cations (Mg²⁺, Ca²⁺, Mn²⁺).

Preparation of PCR Products for Capillary Sequencing:

As with plasmid DNA templates, PCR products should be of sufficient quality to ensure successful sequencing. It is essential to remove potentially interfering substances that may remain in solution after the PCR reaction. Potential contaminants include excess PCR primers, nucleotides, enzyme and buffer components from the PCR reaction as well as unspecific amplification products. These contaminants can sometimes participate, and thus interfere, in the cycle sequencing reaction and lead to poor quality data or no data at all.

Please consider the following recommendations when preparing your PCR reaction(s) for submission:

• All PCR primers must be removed prior to sequencing. Sequencing uses only one primer - instead of the two you used in the PCR reaction. If both forward and reverse PCR primers remain in the PCR solution, they will both act as sequencing primers and anneal to complementary strands with different nucleotide composition, and thus will result in two sequences superimposed on each other that are not readable ("mixed sequence").
• Excess dNTPs must also be removed prior to sequencing as they will upset the specific ratios of dNTPs/ddNTPs required for optimal extension and termination in the cycle sequencing reaction. Please note: While there are many commercial PCR cleanup kits available, we prefer to not recommend specific products.

• It is very important that the PCR reaction is specific and strong! Dependent on the selected experimental parameters, a PCR reaction might yield a single specific amplicon or multiple products. If the PCR product is a smear on an Agarose gel, or more than one band is present, the likelihood of obtaining good sequence data is low. Therefore, we strongly recommend that you analyze a small aliquot of the completed PCR reaction on an Agarose gel to assess its quality. If you have a single specific band, then it will be sufficient to remove all residual PCR primers and unincorporated nucleotides, enzyme and other reaction components. If your PCR reaction generates multiple bands, an excessive amount of primer-dimers or low-intensity smearing, it will be necessary to purify the PCR product of interest via Agarose gel electrophoresis. Alternatively, you may want to spend some time optimizing your PCR reaction to eliminate the presence of additional bands or artifacts and thus save yourself some downstream time.
• Because PCR is intrinsically an exponential process and is usually carried well beyond completion, even rather poor PCR primers will produce amplification in a PCR reaction. Cycle sequencing, however, is strictly linear, and is much more sensitive to inefficient primers. If the PCR primer(s) will also be the sequencing primer(s), make sure they are appropriately designed for automated sequencing. You may be able to adjust your PCR conditions to optimize reactions, but we unfortunately cannot do this. Complete information on proper primer design for Sanger DNA Sequencing can be found here.
• Typical laboratory spectrophotometers cannot with any accuracy measure the small amount of DNA that a PCR reaction generates. Unless you own one of the newer micro-spectrophotometers ("NanoDrop" or similar), you should simply use an analytical Agarose gel to estimate the concentration of your templates. Please compare them to a reference DNA fragment of similar size and known concentration (see DNA Quantitation paragraph above)!
• You should always quantitate your PCR product after purification as no cleanup method will give 100% recovery.