Experimental double deoxy sequencing reaction with T7 phage DNA polymerase
Experimental double deoxy sequencing reaction with T7 phage DNA polymerase
This protocol is modeled after the method of Tabor and Richardson (1987a, 1989b, 1990), which uses a single-stranded DNA template for DNA sequencing (for single-stranded M13 phage and plasmid DNA preparations, see Schemes 4 and 8 in Chapter 3 and Scheme 1 in Chapter 12). This experiment is from the next volume of the Laboratory Guide to Molecular Cloning (3rd edition) by J. Sambrook D.W. Russell.
Operation method
Experimental double deoxy sequencing reaction with T7 phage DNA polymerase
Materials and Instruments
Deionized distilled water Dithiothreitol dNTP and ddNTP Labeling Mix and ddNTP Extension Termination Mix Formamide Sampling Buffer Sequencing Enzyme Dilution Buffer Sequencing Enzyme Reaction Buffer with MgCl2 Sequencing Enzyme Yeast Inorganic Pyrophosphatase Oligonucleotide Primers Template DNA Radioactive Compounds Move makings For more product details, please visit Aladdin Scientific website.
Centrifuges and heads Microtubes or microtitre plates
Retarder fluids and solutions
Deionized distilled water (ice pre-cooled)
Dithiothreitol (DTT) (100 mmol/L)
dNTP and ddNTP, dNTP (0.5 mmol/L) and ddNTP (0.5 mmol/L) reservoirs
5X labeling mix and ddNTP extension/termination mix (ddCTP, ddTTP, ddATP, ddGTP)
These reaction mixes can be prepared by mixing the solution volumes given in Table 12-5.
Formamide Sampling Buffer
Sequencing Enzyme Dilution Buffer
10 mmol/L Tris-Cl(pH7.5)
5 mmol/L DTT
0.5 mg/ml bovine blood albumin
-20°C storage
5X Sequencing Enzyme Reaction Buffer with MgCl2
200 mmol/L Tris-Cl (pH 7.5)
100 mmol/L
125 mmol/LNaCl
-20°C Storage 
5x Sequencing Enzyme Reaction Buffer with MnCl2
200 mmol/L Tris-Cl (pH 7.5)
25 mmol/LMnCl2
125 mmol/LNaCl
-20°C Storage
The presence of Mn2+ in the sequencing enzyme reaction buffer increases the efficiency with which the sequencing enzyme utilizes ddNTP, even in the presence of Mg2+ (TaborandRichardsonlSS%). The result is an efficient termination near the primer in the sequencing reaction, which in turn enhances the intensity of the bands near the oligonucleotide primer or facilitates the elimination of the effects of GC-rich region sequences when dITP base analogs are used.
To use Mn2+, 1ul of 5x sequencing enzyme reaction buffer containing MnCl2 can be added to the labeling reaction prior to addition to sequencing (Fuller et al. 1996) or 0.01 v/v of 1mol/L MnCI2 can be added to the dideoxy-dNTP tinting/termination mix (Kristmse et al. 1990). Manganese-containing buffers that are yellowish or produce a yellow-brown precipitate cannot be used.
TE (pH 7.6)
Enzymes and buffers
Sequencing enzymes (version 2.0)
Sequencing enzyme is supplied by the manufacturer at a concentration of 13 u/ul (approximately lmg/ml). Store at -20°C (do not use frost-free refrigerator).
Yeast Inorganic Pyrophosphatase
Optional, see step 6 The enzyme is supplied by the manufacturer (USB/Amersham Life Science) at a concentration of 5u/ml, which catalyzes the hydrolysis of pyrophosphate to 2 molecules of orthophosphate.
The pyrophosphatase is mixed with the sequencing enzyme in a 1:3 ratio, preferably by mixing the two enzymes in equal volumes and diluting the mixture with 6 times the volume of enzyme dilution buffer. The working concentration of the sequencing enzyme is 1.6 u/ul, and sufficient pyrophosphatase is added to prevent the accumulation of pyrophosphate. 2 ul of diluted enzyme mixture is required for each sequencing reaction.
Nucleic acids and oligos
Oligonucleotide Primers
Concentration is 0.5 pmol/ul (~3.3 ng/ul) in water or TE (pH 7.6).
See the information section "DNA Sequencing Oligonucleotide Primer Reservoir Preparation". For some general primers that bind to vector sequences upstream of the target region, see "General Primers" in Chapter 8.
Template DNA (lug/ul) in TE (PH7.6)
Each set of sequencing reactions requires 1ug of single-stranded DNA or 2.0ug of denatured double-stranded plasmid DNA. double-stranded linear DNA needs to be denatured and complexed with the primers. This means mixing the natural template DNA with an excess of primer, heating in a boiling water bath for about 2 min, and then inserting the tube into an ice water bath. Do not allow the mixture to warm up and use it immediately.
Small-scale preparations of M13 phage recombinant in single-stranded DNA are generally in the range of 0.05-0.5ug/ml. This depends on the growth rate of the particular phage. Under normal sequencing reaction conditions, there is an excess of template DNA. Therefore, small variations in the amount of template per sequencing reaction do not affect the quality of the sequencing results. See Table 12-6 at the end of this program.
Radioactive Compounds
[ a-35S ]dATF (1000Ci/mmol, 10mCi/ml) or
[ a-33P ]dATP(1000~3000Ci/mml, about 20mCi/ml) or
[ a-32P ]dATP(1000Ci/mmol, 10mCi/ml)
If radiolabeled [32P ]dATP is not used as an internal marker, a 35P-labeled oligonucleotide primer at the 5' end can be used for the sequencing reaction. In this case, 2ul of radiolabeled primer (approx. 5X105cpm; approx. 0.5ng) and 2ul of water can be used to replace the unlabeled primer (step 1) and the radiolabeled dATP (step 7) in the reaction, and the other steps are the same. The transfer of [ γ-32P ] from ATP to the 5' end of the oligonucleotide is generally catalyzed by polynucleotide kinase. See Scheme 2 in Chapter 10 for details.
Centrifuge and rotor head
Centrifugal heads or adapters for 0.5 ml microtubes, as well as flattened heads and microtitre plate holders (e.g. Sorvall products) with polystyrene foam or rubber liners.
Specialized equipment
Microcentrifuge tubes (0.5 ml) or microtitre plates (flexible, heat-resistant, U-bottomed 96-well plates with approx. 300ul capacity per well)
See "Microtitre plates" in the information section.
Water baths or heating plates that can be heated to 37°C and 65°C.
Methods
Important: When the sequenced double-stranded plasmid DNA has been denatured and annealed with primers (Scheme 2), the first two steps of this scheme can be ignored. Start directly from step 3.
1. Add to a 0.5 ml microcentrifuge tube or microtitre plate well:
Single-stranded template DNA (1ug/ul) 1ul
Oligonucleotide primer (~1ng/ul) 3ul
5X Sequencing Enzyme Reaction Buffer 2ul
Containing MgCl2 or MnCl2
Water to final volume 10ul
2. Close the tube and incubate at 65°C for 2 min, then remove the tube and allow it to cool to room temperature within 3-5 min.
Some experimenters prefer to use a small heating plate or a beaker filled with water to allow the annealing reaction to cool slowly over the course of 30 min. In our experiments, we have found that the results of these two methods are the same.
3. While the primers and templates are cooling, melt the 5X labeling mix, the ddNTP extension/termination mix, and the radiolabeled dATP, and place them on ice after melting. Annealed boards and primers can be stored at -20°C for several months if necessary.
4. Add 2.5 ul of each ddNTP Extension/Termination Mix to the wells of each 0.5 ml microcentrifuge tube or microtitre plate color-coded or labeled with the letters C, T, A, or G.
When a droplet hangs on the wall of the centrifuge tube or the well of the microburette, it should be centrifuged to the bottom. It should be centrifuged and allowed to settle to the bottom.
5. Dilute the 5x Labeling Mix 5-fold with ice pre-cooled water, requiring 2ul of diluted Labeling Mix for each sequencing reaction.
6. Dilute the sequencing enzyme with ice pre-cooled Sequencing Enzyme Dilution Buffer, with or without yeast pyrophosphatase as described in the materials.
Sequencing enzymes (3 units) are required for each template sequencing. Sequencing enzymes should always be placed on ice. 
7. To perform the labeling reaction, add the following solution to the 10ul of annealing reaction solution from step 2.
Diluted Labeling Mix (from Step 5) 2ul
0.1mol/L dithiothreitol 1ul
[ a-33P ]dATP, [ a-32P ]dATP or [ a-35S ]dATP 0.5ul
Diluted sequencing enzyme (approx. 1.6u/ul) 2.0ul
Gently tap the wall of the centrifuge tube or titer plate to mix the reaction, then incubate at 20°C for 2-5 min.
Diluted sequencing should be kept on ice and not allowed to come to room temperature. Enzyme concentrates provided by the supplier should be stored at -20°C and will be inactivated if left on ice for several hours.
8. When the labeling reaction is nearly complete, the centrifuge tube or microtitre plate should be pre-warmed at 37°C in order to terminate the reaction. This step is important. Then, transfer 3.5ul of labeling solution to the wall of a pre-warmed, labeled microcentrifuge tube or microtitre plate to which an appropriate amount of Dideoxy Mix (step 4 above) has been added.
9. Place the microcentrifuge tubes in a microcentrifuge (with a rotary or coupling head suitable for 0.5 ml tubes or in uncapped 1.5 ml tubes) or the microtitre plate in a centrifuge with appropriate coupling head at room temperature and centrifuge the C, T, A, G tubes or plates at 2000 r/min for a few seconds to mix the mixture. The reaction is then immediately placed in a 37°C heating plate or water bath for 3-5 min.
10. Terminate the reaction by adding 4ul of Formamide Sampling Buffer.
11. The reactions can be stored at -20°C for up to 5 days and can also be analyzed directly by denaturing gel electrophoresis (see protocols 8, 9, or 10, 11 and 12). After thermal denaturation (100°C, 2 min), cool rapidly on ice. Add 3ul of each of the C, T, A and G reactants to the wells of the sequencing gel. 
