How to Determine if a Sequencing Template Meets Quality Requirements

Isolation of plasmid DNA in preparation for automated Sanger sequencing has been simplified by the development of commercial kits. The quality of the final sample is generally tested by spectrophotometer measuring absorbance at wavelengths 260 nm and 280 nm. A ratio value of 1.8 calculated for absorbance measurements at 260 nm by 280 nm indicates the DNA sample is good. However, 260 nm and 280 nm absorbance values do not provide a complete profile of whether DNA preparations meet necessary quality standards for Sanger sequencing applications. Samples may still fail. There are a number of reasons why sequencing reactions are not successful. Two well-known techniques eliminate template quality as a reason for failure.

Scanning Spectrophotometry Provides Important Information about Template Quality

Nanodrop technology has the advantage of highly sensitive spectrophotometric scanning of DNA samples using just 1 ul of volume. Most spectrophotometers available today are capable of performing a wavelength absorbance scan in the ultra violet spectrum. DNA is scanned between wavelengths ranging from 220 nm to 320 nm. The scan profile for quality DNA is shown in figure 1.

A typical DNA profile is a Gaussian (bell-shaped) curve with the maximum peak height absorbing at 260 nm. A secondary peak will also begin to form around 220 nm. Deviations in the shape of either curve could indicate the presence of salts or other contaminants.

Figure 2 provides a profile of plasmid DNA containing salts compared to clean DNA. The Gaussian curve should drop almost to the baseline when DNA is free of contaminating salts in the range 230 nm to 240 nm. Salts present in the sample will absorb wavelengths in this range. Excessive amounts of salt in DNA samples may show little or no dip in the curve between 220 nm and 260 nm.

Figure 3 represents a profile of DNA that contains either protein (DNA from tissue) or phenol (plasmid DNA). Phenol residue in DNA can result from phenol/ chloroform extraction (a method of extraction that was widely used prior to the development of commercial kits). Despite the availability of kit-based methods, many researchers opt to use phenol/ chloroform instead. It is important to remove phenol residues with ethanol precipitation in the final purification step. Phenol residue can negatively affect the PCR amplification in Sanger sequencing.

Scanning spectrophotometry successfully identifies salt contaminants in plasmid DNA and DNA samples amplified in the Polymerase Chain Reaction (PCR). PCR fragments will have a lower concentration. But purified PCR products should still show a scan similar to plasmid preparations.

Agarose Gel Electrophoresis Identifies Nicked DNA

Once plasmid DNA has been determined to be free from salt contaminants it should work well for Sanger sequencing. However, this is not always the end result. Scanning spectrophotometry does not reveal whether DNA is supercoiled or nicked as described previously (Nicked Plasmid DNA Prevents Automated Sanger Sequencing). Nicked DNA has been damaged mechanically. Aggressive vortexing during DNA purification is one cause of DNA damage. Nicked DNA does not amplify in Sanger sequencing applications because the double stranded helix does not maintain a tight formation. Taq polymerase is unable to fulfill a lock-key attachment to the DNA and catalyze extension. For automated sequencing to work, plasmid DNA must maintain a supercoiled structure.

Agarose gel electrophoresis successfully separates nicked DNA (slow migrating) and supercoiled (fast migrating) as shown in figure 4. Nicked DNA moves more slowly through the gel because it is a larger molecule than supercoiled DNA. It should be noted that plasmid preparations typically have a mixture of nicked and supercoiled DNA. Presence of some nicked DNA should not cause concern. It is the lack of supercoiled DNA that causes sequencing failures.

Scanning spectrophotometry and agarose gel electrophoresis combined provide a good assessment of whether template DNA purification has been successful. However, it does not guarantee Sanger sequencing will be successful every time. Primer selection, GC content and presence of poly base regions and hairpins could also affect sequencing. Problems of this nature are related more to base content and not preparation of the sample.

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Preparing Plasmid DNA for Automated Sanger Sequencing

Cesium chloride and phenyl chloroform were two early methods used for isolating a sample of DNA from a bacterial vector. Cesium chloride produced a sample that was very clean. However, the method was labor intensive. Phenyl chloroform was less labor intensive, but phenyl contamination was difficult to remove even using the final alcohol precipitation step. Fortunately, commercial kits were developed around the same time Sanger sequencing became automated. Presently there are numerous commercial kits from which a researcher could choose.

However, using a commercial kit does not guarantee that a resulting DNA sample will be clean and free from contaminants. The final step of the sample preparation often includes Tris or another buffer (salt) for elution. Other times the DNA may incur physical damage and become nicked. Preparing a sample for automated sequencing includes following the recommended guidelines provided for the commercial kit. However, low yields can require additional steps to concentrate the sample. Simple drying methods concentrate everything in the sample including buffer salts.

Steps Involved in Commercial DNA Preparation Kits

The general protocol for commercial kits used to isolate purified DNA from plasmids is fairly similar regardless of the kit. After plasmid bacteria have been grown overnight in liquid media, the cells are pelleted by centrifugation into a solid mass. The media is then discarded before beginning the kit provided procedure (Figure 1).

The pellet of cells is resuspended in buffer and transferred into a 1.5 or 2 ml tube (Step 1). Lysis reagent is added (Step 2). The lysis reagent disrupts the bacterial cell membranes freeing internal components. The lysis buffer is neutralized with neutralizing solution and cell components form a precipitate with the DNA still in solution (Step 3). The resulting solution is centrifuged to pellet the cell waste leaving the DNA in solution.

DNA is captured on a filter or resin by transferring the DNA solution to a collection vessel (Step 4). Excess liquid is pulled through the filter by centrifugation or vacuum filtration. The remaining DNA on the filter is then washed removing any cell waste left (step 5). The DNA is then collected in a clean tube using water or elution buffer (step 6). Elution buffer is provided in the commercial kits. It is comprised of Tris buffer in water. Fortunately, manufacturers no longer include EDTA in elution buffer. EDTA interferes with magnesium chloride, a necessary component in any PCR amplification. Water is the preferred media for DNA to be used for Sanger sequencing as no salts are being introduced to the sample.

The process may take as little as 15 minutes after bacterial cells are grown overnight. The final DNA sample is relatively pure and clean from other cellular debris that could interfere with Sanger sequencing.

Quality Testing of the DNA Sample

DNA sample quality can be determined using two simple methods. The methods will be discussed in detail in the next article, but are summarized below.

Scanning spectrophotometry using ultraviolet wavelengths between 220 nm and 310 nm provides a general profile of the overall quality of the DNA (Figure 2). DNA absorbs light in the range of 240 nm to 300 nm with the maximum peak at 260 nm. Absorbance at 260 nm is also used to calculate the concentration of the DNA. Another factor often used when measuring DNA quality is calculating the 260 nm / 280 nm absorbance ratio. A good value for this ratio is 1.8 to 1.9. Ratio values below 1.6 indicate the DNA sample contains contaminants.

A good scanning profile does not always prove the DNA is good. Nicked DNA cannot be identified by scanning and may inhibit quality Sanger sequencing. (https://agctsequencing.wordpress.com/2012/02/16/nicked-plasmid-dna-prevents-automated-sanger-sequencing/)

Agarose gel electrophoresis will separate DNA into bands indicating whether the DNA has remained supercoiled.  This supercoiled DNA is required for sequencing. It will also show if the DNA has been damaged and the supercoil has loosened when the DNA is nicked. Nicked DNA migrates more slowly through an agarose gel and will separate from the supercoiled DNA.

Both quality tests provide necessary data to show whether the final DNA sample is clean and of high quality.

Additional Concentration Required

Once the DNA sample has been isolated and appears clean using test procedures, it may be necessary to adjust the concentration to meet submission guidelines. Samples with a concentration higher than required are diluted in water. Although some buffer is still present, the buffer is diluted along with the DNA. Generally dilute buffers (without EDTA) do not interfere with amplification for Sanger sequencing. What if the sample needs to be more concentrated?

Drying a sample in elution buffer is not an effective means for concentrating a sample. Ethanol precipitation is a preferred method because salts are removed along with excess water. Scanning spectrophotometry is an effective means of quantifying the DNA.

What is most important for researchers to remember when isolating plasmid DNA with a commercial kit is to thoroughly read the directions. The kits use enzymes to disrupt the bacterial cell membrane and remove components such as RNA. Enzymes are fragile. Excess shaking or vortexing could damage the enzymes. Commercial kits often include precautions to use care when working with these important enzymes.

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