Method: Sequencing of Double Stranded DNA Using Dideoxy Chain Termination

May 10, 1990

Terry C. Lairmore

Principle:

This method is used to sequence double stranded DNA, such as a plasmid insert or purified PCR product. The dideoxy chain termination (or enzymatic) method of DNA sequencing involves the in vitro synthesis of a DNA strand by a modified bacteriophage T7 DNA polymerase (SequenaseR/USB) using a single stranded DNA template. Synthesis is initiated at a specificsite on the sequencing template, as determined by the annealed sequencing oligonucleotide primer. The synthesis reaction is terminated by the incorporation of a dideoxynucleotide analog (ddNTP) that will not accept further elongation of the synthesized strand. Four separate reactions are set up for each template to be sequenced, each containing one nucleotide as the ddNTP plus the other three deoxynucleotides (dNTPs). For each reaction, enzyme catalyzed synthesis will be terminated in a fraction of the population of chains at each site where the ddNTP is incorporated (e.g. in the ddGTP reaction tube, a population of chains result with a fraction of the chains terminated at each site a "G" occurs). Radiolabeled dATP is included in the synthesis, and the products are size separated by high resolution denaturing polyacrylamide gel electrophoresis and visualized by autoradiography. The Sequenase kit also includes the reagents for sequencing reactions using ddITP (in place of ddGTP). This set of reactions is used only when you wish to attempt to resolve a problem with a compression (i. e., when the secondary structure of the DNA interferes with the size separation in the sequencing gel).

Time required:

2-4 Days

• Pouring of sequencing gel: 1-2 hours, polymerization 1 hour to overnight
• Sequencing reactions: 1 hour and 30 minutes (with experience)
• High-resolution denaturing polyacrylamide gel electrophoresis: 4-8 hours
• Removal of Urea and Gel Drying: 1 hour 30 minutes
• Autoradiography: overnight to 2 days

Special reagents:

All of the following required reagents are included in the SequenaseR kit (USB):

• Sequenase 5X buffer (200 mM Tris-HCl, 100 mM MgCl2, 250 mM NaCl)
• Dithiothreitol (DTT) 0.1 M
• Labeling Mix
• Termination Mixes (for ddGTP, ddATP, ddTTP, ddCTP)
• SequenaseR enzyme
• Enzyme dilution buffer (10 mMTris-HCl pH 7.5, 5 mM DTT, 0.5 mg/ml BSA)
• Stop solution (95% formamide, 20 mM EDTA, 0.05% Bromophenol blue, 0.05% Xylene cyanol FF

Special Equipment:

• Microcapillary flat pipet tips (Midwest Scientific, #MPT1000F)
• Baserunner sequencing gel unit (IBI)
• High voltage power supply

  Gel Dryer

Description of Reactions:

It is important to understand the reactions occuring at each step in order to perform several sequencing reactions simultaneously because the time interval allowed for each step is critical for certain steps. The first step is the labeling step which incorporates the 32P-dATP. In this step the primer is extended using limiting concentrations of dNTPs including 32P-dATP. This reaction should be allowed to continue at 4 degrees C for 3-5 minutes only , to result in labeled chains limited in length from several nucleotides to hundreds of nucleotides. In the second step, higher concentrations of 3 of the dNTPs plus the remaining nucleotide triphosphate as a dideoxy derivative results in termination of each of the growing chains at the next site where the ddNTP can be incorporated. This reaction is carried out at 37 degrees C and can continue for 5 minutes or longer.

Procedure:

The sequencing protocol is accomplished by pouring the polyacrylamide gel on Day 1, allowing the gel to polymerize and the gel is stored overnight at room temperature wrapped in cellophane. The sequencing reactions may also be prepared on Day 1 and frozen at -20 degrees C, then loaded on the sequencing gel the following day.

Day 1

Pouring of Sequencing (8% polyacrylamide/urea) denaturing gel

This is probably the most difficult part of sequencing, and it is recommended that taping the plates and pouring of the plug and gel be learned from an individual with experience.

Wash plates with mild soap and a non-abrasive sponge, rinse with dH20 and clean with 100% EtOH. Siliconize only the mirrored plate with a light coat of SigmacoteR in the fume hood using a Kimwipe and a light circular motion (wear gloves) and quickly assemble plates with 0.4 mm spacers before any dust collects. Tape the bottom of the plates securely with water-proof tape and clamp sides (Do not clamp the bottom).

Recipe for 8% polyacrylamide sequencing gel:

75 ml for two 45 cm plates or one 60 cm plate:	50 ml for one 45 cm plate:
31.5 g urea	21 g urea
31 ml ddH20	21 ml ddH20
7.5 ml 10X TBE	5 ml 10X TBE
15 ml 40% acrylamide/ 2% bis stock	10 ml 40% stock
375 µl 10% ammonium persulfate (APS)	250 µl 10% APS
Immediately before pouring, add:
35.0 µl TEMED	24.0 µl TEMED

The urea should be weighed out first and allowed to dissolve in the water with the aid of a magnetic stirring bar (do not use heat) while the plates are cleaned and prepared. Add the remaining ingredients, filter (0.45 micron), and leave the acrylamide solution on ice in the disposible filter container. It is advisable to de-gas the acrylamide solution by gently swirling with the vacuum on until no more bubbles are produced.

A separate plug should be made (4 ml of above acrylamide solution and 8 µl of TEMED), poured down the side of the gel and allowed to completely solidify before attempting the pour the entire gel. The TEMED should not be added to the acrylamide until you are completely ready to pour the gel. Hold the gel cast at an incline and pour the gel carefully but quickly, taking care not to trap bubbles. This is accomplished using a 60 cc syringe. It may be necessary to lightly tap the outside of the plate or alter the incline to remove bubbles. After the full length of the gel is poured, carefully insert the comb (shark's tooth or standard), place in a horizontal position and allow to polymerize at least one hour. The gel may be wrapped in cellophane and stored overnight. (Longer storage is not recommended).

Sequencing Reactions (example below is for sequencing the insert in a plasmid such as pUC or Bluescript using the universal and reverse primers)

1. Prepare plasmid DNA as described elsewhere in the alkaline lysis miniprep protocol. Resuspend the pellet in 50 µl ddH20. Pipet an 18 µl aliquot of this into a clean eppendorf tube for sequencing.
2. Add 2 µl of 2 M NaOH/ 2mM EDTA. Incubate at room temperature for 20 minutes (alkaline denaturation).
3. Add 2 µl ammonium acetate (pH 4.5) and then 50 µl of 95% EtOH, mix well and place on ice for 15 minutes (precipitation).
4. Spin in microcentrifuge at 4 degrees C for 10-15 minutes and carefully decant the supernatant.
5. Wash pellet with 70% EtOH, respin, and decant supernatant. Invert the tube on a Kimwipe to air dry.

The sequencing reactions may now be carried out using the SequenaseR kit and a modification of the double stranded sequencing protocol: The 4 reaction tubes (G, A, T, C)should be labeled and the appropriate termination mix (3.5 µl) dispensed in each before beginning the protocol. Be sure to choose the ddGTP labeling mix unless you intend to use the ddITP reactions for resolving compressions.

1. Resuspend the pellet in:

   • 6 µl ddH20
   • 2 µl 5X Sequencing buffer
   • 2 µl Sequencing primer (0.5 pmol/µl)
   • or, 2 µl 5X Sequencing buffer, 0.5-1.0 pmol primer, and the appropriate amount of water to a total volume of 10 µl.

2. Incubate at 37 degrees C for 15 minutes for annealing of primer. (Alternatively, the tube may be heated to 65 degrees C for 2 minutes and allowed to cool to room temperature over 30 minutes. This has not been found to be necessary in my experience if the alkaline denaturation step is used).
3. Add 2 µl diluted labeling mix 1:5 to 1:15. (Note: for sequencing within 30 bases of primer, dilution should be 15 fold)
   • 1 µl 100 mM DTT
   • 1 µl 32P-dATP
   • 2 µl diluted Sequenase (diluted 1:8 in ice-cold TE immediately before use).

   Allow reaction to extend at room temperature (or at 4 degrees C) for 3-5 minutes.

4. Dispense 3.5 µl of the above mixture into each of 4 prelabeled tubes (G, A, T, C) containing 2.5 µl of appropriate termination mix (ddGTP, ddATP, ddTTP, ddCTP) and incubate at 37 degrees C for 5 minutes or longer.
5. Add 4 µl of Sequenase stop solution to each tube. Samples may be stored at -20 degrees C until loaded on the sequencing gel.

Remove the sequencing gel from refrigerator, place on Baserunner (IBI), and attach buffer chambers. After filling the buffer chambers with 1X TBE, gently flush out the wells with a syringe and needle and prerun for 30 minutes to 1 hour.
1. Thaw the sequencing reaction samples, heat to 75-80 degrees C for 2 minutes and immediately load 1-3 µl from per lane. Use the specialized microcapillary flat pipet tips (Midwest Scientific, #MPT1000F) to load the gel. Slowly layer each sample into the bottom of each well, avoiding bubbles.
2. The lanes should be loaded G, A, T, C in adjacent order for each sequencing reaction. Run at a constant power of 55 watts, with voltage from 1800 to 2000 V or greater. A second aliquot of the samples may be heated again and reloaded after a variable period of time in order to read further. Ideally, an area of overlap between the bottom of the first load (larger fragments which have run longer) and the top of the second load (smaller fragments which have run less time) will be found on the autoradiogram. (One recommendation is to run the first load twice the amount of time it takes the bromophenol blue to run off, and the second load 2-2.5 hrs).
3. At the completion of the run, turn off power supply, carefully drain buffer chambers (buffer must go in liquid radioactive waste container), and remove the gel from the apparatus.
4. Place the gel with the glass plate face down on a flat surface. Remove the mirrored glass plate (the gel should stick to the unsiliconized glass plate). Leave the spacers in place on each side.
5. Carefully soak the gel for 10-15 minutes in 10% glacial acetic acid on the glass plate with minimal rocking (be careful that the gel doesn't float off the glass plate).
6. Remove the gel from the acetic acid. Cut two pieces of Whatman paper the size of the glass plate and carefully place on top of the gel with glass plate below. Turn the Whatman paper, gel and glass plate over as a unit and then remove the glass plate (the gel sticks to the Whatman). Cover the gel with Saran wrap and dry in a gel dryer for 1-1.5 hours.
7. Expose film overnight (usually sufficient for 32-P(Potassium isotope 32)) or longer.

Reagents:

• Sigmacote (Sigma, #SL-2)
• Acrylamide (electrophoresis grade)
• N,N'-Methylenebisacrylamide (electrophoresis grade, Ultra-Pure/BRL, #5516UB)
• Ammonium persulfate (Ultra-Pure/BRL, #5523UA)
• TEMED (N,N,N'N' Tetramethylethylenediamine, Ultra-Pure/ BRL, #5524UB)

Recipes for Acrylamide stocks:

• 40% Acrylamide/ 2% bis stock

  acrylamide:	38 g
  N,N'-methylene bisacrylamide:	2 g
  dH20:	to 100 ml

  Safety Considerations: Acrylamide is a neurotoxin (especially in solid form). Work with this substance should be done in the hood. Wear gloves and a mask when weighing out and handling. Clean up all spills immediately.

References:

Tabor, S. and C. C.Richardson. (1987) "DNA sequence analysis with a modified bacteriophage T7 DNA polymerase." Proc. Nat. Acad. Sci. USA. 84: 4767-4771.

Sanger, F. Miklen, S. and A. R. Coulson. (1977) "DNA sequencing with chain-terminating inhibitor." Proc. Nat. Acad. Sci. USA 74: 5463-5467.

SequenaseR Step by Step Protocols (1989) United States Biochemical Corporation.