13.9: Lab Technique - DNA Sequencing

Preparation of the DNA template for sequencing

The DNA region to be sequenced must be prepared prior to sequencing. In the most common scenario, the DNA is amplified through PCR to add unique restriction sites to the ends of the DNA. This DNA is then cloned into a sequencing plasmid (Figure \(\PageIndex{1}\)). Sequencing plasmids have a multi-cloning site (or polylinker) made up of multiple restriction sites and allow for directional cloning into the plasmid. In many sequencing primers, the multi-cloning site is flanked by several sequencing primer sites, giving the researcher flexibility in what sequencing primer will be used in the sequencing reaction. Examples of common sequencing primer sites include sequences from the M13 and T7 bacteriophage. For more information about cloning into a plasmid, refer to Chapter 13.5 Lab Technique: Ligation of a DNA Insert into a Plasmid.

Once cloning is complete, the recombinant sequencing plasmid must be purified to remove contaminants, like salt and cloning enzymes, that can interfere with the sequencing reaction. Numerous companies offer kits that will remove these contaminants and produce a recombinant sequencing plasmid of high purity. Purification of the sample after the sequencing reaction has been completed can also be performed with commercial kits in order to improve output by the DNA sequencer.

Components of the Sanger sequencing reaction (i.e., the Sanger reaction)

1. Template DNA:
   • cloned into a sequencing primer
   • purified to remove contaminants that could interfere with sequencing
2. A DNA polymerase:
   • usually the large subunit of the DNA polymerase I complex called the Klenow fragment
   • Klenow fragment contains the polymerization site and the site for proofreading
   • Klenow ensures the DNA region is sequenced without major errors
   • Taq polymerase may lead to the sequencing outside of the desired DNA region
3. A sequencing primer:
   • a short piece of single-stranded DNA that binds to the template DNA
   • acts as a "starter" for polymerase extension
   • often sold in a set as a forward sequencing primer that binds the "anti-sense" DNA strand and a "reverse" sequencing primer that binds the "sense" DNA strand
   • sequencing using two primers ensures coverage of the DNA region you wish to sequence
4. A dNTP mix:
   • dATP, dTTP, dCTP, dGTP
   • added to the sequencing reaction in excess amounts
5. A ddNTP mix:
   • a mixture of dideoxy, or chain-terminating, versions of all four nucleotides (ddATP, ddTTP, ddCTP, ddGTP)
   • added to the sequencing reaction in small amounts
   • each labeled with a different color of dye
6. Other components:
   • polymerase buffer
   • MgCl₂

Today, most companies will sell a sequencing "master mix" containing each of these components with the exception of the sequencing primer and the DNA template.

Overview of the Sanger Reaction

The DNA sample to be sequenced is added to a PCR tube along with the other components of the sequencing reaction and subject to a PCR program made up of multiple cycles of denaturing, annealing, and extension. For more information about the PCR protocol, refer to Chapter 13.2 Lab Technique: Polymerase Chain Reaction (PCR). The first temperature of the cycle is a denaturing step in which the sample is heated to high temperatures (i.e., 95°C) in order to denature the double-stranded template DNA. The second step of the cycle is an annealing step where the reaction temperature is lowered to an "annealing" temperature (usually between 50°C and 55°C) so that the sequencing primer can bind to the single-stranded "anti-sense" template. The temperature is raised again, in the last step of the cycle, to an extension temperature (i.e., 60°C) in which the DNA polymerase extends from the sequencing primer and synthesizes the daughter DNA strand. Because the dNTPs are added in excess in a sequencing reaction, the DNA polymerase will synthesize the majority of the daughter DNA strand using these nucleotides. However, the DNA polymerase will eventually incorporate a ddNTP into the growing DNA strand. When this happens, the extension of the daughter DNA strand stops. This PCR cycle is repeated in a number of times and by time the PCR program is complete, it is virtually guaranteed that a ddNTP will have been incorporated at every single position of the daughter DNA strand. The DNA sample that results from this PCR program will contain fragments of different lengths, ending at each of the nucleotide positions in the original DNA (Figure \(\PageIndex{2}\)).

While individual laboratories often prepare the DNA sample for sequencing and perform the Sanger reaction, it is unusual for them to run the DNA sample through the DNA sequencer. This is because DNA sequencers are very expensive. As a result, sequence analysis of the sample is usually done in a sequencing facility by trained laboratory technicians. These facilities will run the DNA sample through their sequencer once the Sanger reaction has been completed and will forward a compiled sequence to the laboratory. Many of these facilities also offer a "full-service" option where Sanger reaction is performed, along with purification of the sample, subsequent analysis by the sequencer. Laboratories can also purchase sequencing and purification kits from the sequencing facility that are compatible with the facility's sequencers.

Lab Protocol: Sanger Sequencing

The protocol given below is for the sequencing of double-stranded DNA (i.e., a sequencing plasmid) using the commercial kit ProDye^TM Terminator Sequencing System from Promega. This commercial kit is compatible with a variety of DNA templates, such as sequencing plasmids and PCR products. The sequencing reaction can be analyzed by numerous DNA sequencers, including those from Applied Biosystems (a division of Thermo Fisher). It is important to confirm compatibility with other DNA sequencers. Similar commercial kits from other companies may be used for other DNA sequencers.

1. Clone the DNA region to be sequenced into a suitable sequencing plasmid, such as pUC19.
2. Purify the resulting plasmid to remove contaminants.
3. Determine the concentration of the plasmid and its purity using a UV spectrophotometer.
4. Add 300 to 500 ng of plasmid DNA to a PCR tube. Note that different DNA quantities will be added if sequencing single-stranded DNA or PCR products.
5. Add 2.5 to 5.0 pmole of sequencing primer to the tube.
6. Add 8.0 µL of the ProDye^TM 2.5X Master Mix to the tube. Beyond this point, protect the tube from light exposure.
7. Add nuclease-free water to a final volume of 20 µL.
8. Place the tube in thermocycler
9. Run the following protocol
   1. 96°C for 1 minute
   2. 96°C for 10 seconds
   3. 50°C for 5 seconds
   4. 60°C for 4 minutes
   5. Repeat Steps 2 through 4 for a total of 25 cycles
   6. Hold at 4°C
10. Place the reaction at 4°C overnight or -20°C for longer-term storage. Protect from light exposure.

Protocol: Ethanol Purification of Sequencing Reaction

Once the Sanger sequencing reaction has been completed, the sample will need to be purified to remove any remaining reaction components that will interfere with the DNA sequencer. Purification can be done by sequencing facilities (for an additional cost) or can be done by the researcher. Numerous commercial kits are available for clean-up of PCR products (e.g., QIAquick PCR purification kit from Qiagen). If cost is a concern, the ethanol purification method given below is easy to perform. However, care must be taken in this procedure not to lose the DNA pellet.

1. Add 5.0 µL of 125 mM EDTA (pH 8.0) and mix well.
2. Add 60 µL of 100% ethanol.
3. Vortex briefly
4. Incubate for 15 minutes at room temperature, protected from the light.
5. Centrifuge at 4°C for 45 minutes at 2000 xg.
6. Decant off the liquid.
7. Add 60 µL of 70% ethanol.
8. Centrifuge at 4°C for 15 minutes at 2000 xg.
9. Decant off the liquid.
10. Centrifuge at 4°C for 1 minute at 2000 xg.
11. Carefully aspirate any remaining liquid and air dry the pellet.
12. Analyze the sample in a DNA sequencer.