In addition to its use in PCR reactions, heat-stabilized DNA polymerase can be used as a replacement for sequencing enzymes or Klenow fragments for double deoxy chain termination sequencing. Its ability to perform isothermal chain termination at elevated temperatures (70°C) reduces problems in sequencing reactions due to mismatches between primer pseudo-binding sites on the template DNA and primer annealing, and it can also be used for sequencing templates enriched in secondary structure. This experiment was derived from the next volume of the Laboratory Guide to Molecular Cloning (Third Edition) by [American] J. Sambrook D.W. Russell.
Operation method
Dideoxy sequencing reaction experiments with Taq DNA polymerase
Materials and Instruments
Deionized distilled water ddNTP dNTP formamide spiking buffer. Enzymes and buffers. taq enzyme or similar heat-stable DNA polymerase Taq enzyme dilution buffer Nucleic acids and oligonucleotides Oligonucleotide primers Template DNA (100ug ul) dissolved in TE Radioactive compounds 5'32P-labeled oligonucleotide primer Move makings For more product details, please visit Aladdin Scientific website.
Microcentrifuge tubes (0.5 ml) or microtitre plates Programmable thermal cycler with multiwell heating plate Water bath heated to 65°C
Buffers and solutions
See Appendix 1 for the composition of reagents, buffers, and reservoirs, and dilute the reservoirs to the appropriate concentration before use.
Deionized distilled water (ice pre-cooled)
ddNTP, 4 ddNTP reservoirs (5 mmoI/L)
dNTP, 4 dNTP reservoirs (lmmol/L)
Formamide Sampling Buffer
10x Labeling Mix and ddNTP Extension/Termination Mix
When the sequencing template is enriched with secondary structures, replace dGTP in the 10X Labeling Mix and ddNTP Extension/Termination Mix with equimolar 7-deamino-2'-dGTP.
Enzymes and Buffers
5x Reaction Buffer
200 mmol/L Tris-Cl (pH 8.8)
25 mmol/LMgCl2
Taq enzyme (5 u/ul) or similar heat-stable DNA polymerase
Taq enzyme dilution buffer
25 mmol/L Tris (pH 8.8)
0.01 mmol/LEDTA (pH 8.0)
0.15% Tween-20
0.15% NP-40
Reaction conditions and reaction buffers for heat stabilized enzymes derived from other organisms may vary slightly. The optimum buffer conditions for different enzymes are given in the manufacturer's instructions. One unit of heat-stabilized DMA polymerase activity is usually defined as the amount of enzyme required to convert 10 nmol of nucleotide to the acid precipitate form in a 30-min reaction at 70-80°C. The amount of enzyme required to convert 10 nmol of nucleotide to the acid precipitate form in a 30-min reaction at 70-80°C is considered to be one unit of enzyme activity.
Nucleic Acids and Oligonucleotides
Oligonucleotide Primers
Concentration of 1.0 pmol/pl (~6.6 ng/ul), dissolved in TE (pH 7.6).
For some general primers that bind to vector sequences upstream of the target region, see "General Primers" in the information section of Chapter 8 and "DNA Sequencing Oligonucleotide Primer Reservoir Preparation" in the information section at the end of this chapter. 
Template DNA (100ug/ul) dissolved in TE (pH 7.6)
Each set of sequencing reactions requires 500ng of single-stranded DNA or 1.0ug of denatured double-stranded plasmid DNA. denature the double-stranded linear DNA and denature it with the primers. To denature the double-stranded linear DNA and complex it with the primer, mix the natural template DNA with an excess of primer, heat in a boiling water bath for about 2 min, and then insert 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 DMA are typically prepared at concentrations in the range of 0.05 to 0.5ug/ml. This depends on the growth rate of the particular phage. In the case of a normal Taq enzyme-catalyzed sequencing reaction, there is an excess of board DMA. Therefore, small variations in the amount of template per sequencing reaction do not affect the quality of the sequencing results. See Solution 4 for troubleshooting.
Radioactive compounds
[ a-32P ]dATP (3000Ci/mmol, 10mCi/ml) or
[ a-35S ]dATP(1000Ci/mmol,10mCi/ml) or
[ a-33P ]dATF(2000-4000Ci/mmol,~10mCi/ml) or
5 '32P-labeled oligonucleotide primers
If radiolabeled dATP is not used as an internal marker, 32P- or 33P-labeled oligonucleotide primers at the 5' end can also be used for sequencing reactions. In this case, 1.0~1.5pmol of radiolabeled primer and 2ul of water can be used to replace the unlabeled primer and radiolabeled dATP in the reaction (step 1), and the other steps are the same. Generally, polynucleotide kinase is used to catalyze the transfer of [ γ-32P ] from ATP to the 5' end of the oligonucleotide. See Scheme 2 in Chapter 10 for details.
Specialized equipment
Microcentrifuge tubes (0.5 ml) or microtitre plates (flexible, heat-resistant, U-well 96-well plates with a capacity of 300ul per well).
See "Microtitre plates" in the information section.
Programmable thermal cyclers with multiwell heating plates (e.g. Dri-Blockcyclers, Techne).
Alternatively, water baths at 45°C and 72°C are available. See step 8 for instructions.
A water bath can be heated to 65°C.
Methods
1. Into the well of a 0.5 ml microcentrifuge tube or microtitre plate add.
Single-stranded template DNA (250fmd) (100ng/ul) 5.0ul
Oligonucleotide primer (0.5 pmol) (approx. 3.3 ng/ul) 3.0ul
5x reaction buffer 20.ul
2. incubate sealed microcentrifuge tubes at 65°C for 2 min. remove the tubes from the water bath and allow them 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 slow the annealing reaction over the course of 30 min. In our experiments, we have found that the results are the same for both methods.
3. While the primers and templates are cooling, melt the 10X Labeling Mix, the ddNTP Extension/Termination Mix, and the radiolabeled dATP, and place on ice after melting.
4. Add 4ul of the corresponding ddNTP Extension/Termination Mix to each color-coded 0.5 ml microcentrifuge tube or each pre-labeled C, T, A, and G microtitre plate well (e.g., 4ul of ddCTP Mix for microcentrifuge tubes and microtitre plate wells labeled C, 4ul of ddTTP Mix for microcentrifuge tubes and microtitre plate wells labeled T, etc.). ) and place the microcentrifuge tube or microtitre plate on ice.
5. Dilute enough heat-stabilized DNA polymerase for all template sequencing at a ratio of 1:8, for example:
TaqDNA polymerase (5~101u/ul)
Enzyme Dilution Buffer 7ul
Each set of four sequencing reactions requires 2ul (2u) of diluted enzyme. The final concentration of the enzyme should be approximately lu/ul. The diluted enzyme should be stored on ice at all times.
6. Add the following to the annealing tube (step 2 above).
10x Labeling Mix 2ul
radiolabeled dATP 0.5ul
Diluted DNA polymerase (~1u/ul) 8ul
Vortex and mix well, then incubate the reaction at 45°C for 5 min.
7. Transfer 4ul of labeled reaction solution to each well of a C, T, A, G tube or microtitre plate containing the appropriate Dideoxy Termination Mix (step 4 above), adding along the wall of the microcentrifuge tube and microtitre plate.
8. Place the small centrifuge tubes in a microcentrifuge (with a suitable rotary head or adapter for 0.5 ml tubes, or into uncapped 1.5 ml centrifuge tubes) or the microtitre plate in a centrifuge with a suitable adapter, and centrifuge tubes or plates C, T, A, and G at 2000 r/min for a few seconds, mixing the reactants. incubate at 72°C for 5 min.
Place the small tubes or plates in a high-efficiency heating block or in water, oil or other medium with high thermal conductivity. The ideal temperature for TaqDNA polymerase will not be reached during such a short incubation if the plate is placed in an empty chamber at 72°C, but the temperature of the plate will not be reached if the plate is incubated at 72°C for 5 min.
9. Terminate the reaction by adding 4ul of Formamide Spiking Buffer.
10. These 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.