Double deoxygenation sequencing using E. coli DNA polymerase I Klenow fragments and single-stranded DNA templates
Double deoxygenation sequencing using E. coli DNA polymerase I Klenow fragments and single-stranded DNA templates
When Sanger and colleagues established the first DNA strand termination extension method, there was only one suitable DNA polymerase available, the Klenow fragment of E. coli DNA polymerase I. It had the activity to polymerize dNTP in the presence of a template, but lacked the 5'-3' exonuclease activity of intact polymerase I. It had the activity to polymerize dNTP in the presence of a template, but lacked the 5'-3' exonuclease activity of the intact polymerase I. The polymerase I fragment was used to polymerize dNTP in the presence of a template. This experiment is from the next volume of the Laboratory Guide to Molecular Cloning (3rd edition) by [American] J. Sambrook D.W. Russell.
Operation method
Double deoxygenation sequencing with E. coli DNA polymerase I Klenow fragments and single-stranded DNA templates
Materials and Instruments
dATP Deionized distilled water EDTA Extension Termination Mix and Tracer Mix Formamide Sampling Buffer Tris-Cl Enzyme and Buffer E. coli DNA Polymerase I Kienow Fragment Nucleic Acids and Oligonucleotides Oligonucleotide Primers Single-stranded DNA Templates Radioactive Compounds Move makings For more product details, please visit Aladdin Scientific website.
Centrifuges and rotary heads Microcentrifuge tubes or microtitre plates
Buffers and solutions
Reagents, buffers, and reservoir solutions are listed in Appendix 1 and are diluted to the appropriate concentration before use.
dATP (0.1 mmol/L)
Optional, see step 4.
Deionized distilled water
EDTA (10 mmol/L, pH 8.0)
Extension/termination mix and tracer mix
These reaction mixes can be prepared by mixing dNTP and ddNTP reservoir solutions, see Table 12-7.
Formamide Sample Buffer
TM Buffer
100 mmol/L Tris-Cl (pH 8.5)
50 mmol/LMgCl2
Tris-Cl(1mol/L,pH8.0)
Enzyme and buffer
E. coli DNA polymeraseⅠKienow fragment (5u/ul)
Approximately 2.5 units of enzyme are required for each set of four dideoxy sequencing reactions. klenow fragments are usually stored in a buffer containing 50% glycerol. Excessive use of the enzyme in sequencing reactions can cause distortion of the sequencing gel due to the interaction of glycerol with borate ions in TBE, the standard buffer for gel preparation and gel electrophoresis (see Troubleshooting at the end of this protocol).
Nucleic Acids and Oligonucleotides
dNTP, ddNTP, dNTP(1 mmol/L), and ddNTP(5 mmol/L) Reservoirs
Oligonucleotide primers
Concentration is about 0.5 pmol/ul (about 3.3 ng/ul), dissolved in water.
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 "Preparation of Oligonucleotide Primer Reservoirs for DNA Sequencing" at the end of this chapter.
Single-stranded DNA templates
Single-stranded DNA template at a concentration of about 0.05 pmol/ul, equivalent to about 0.15ug/ul of M13 phage single-stranded DNA.
Concentrations for small-scale preparations of M13 phage recombinant single-stranded DNA are typically in the range of 0.05 to 0.5ug/ml. This depends on the rate of production 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 Troubleshooting at the end of this program.
Radioactive compounds
[ a-32P ]dATP (3000Ci/mmol, 10mCi/ml) or
[ a-33P ]dATP(3000Ci/mmol,10mCi/ml) or
[ a-35S ]dATF(1000Ci/mmol,10mCi/ml) or
5 '32P-labeled cold nucleotide primers
If radiolabeled dATf is not used as an internal marker, a 32P-labeled oligonucleotide primer at the 5' end can also be used for the sequencing reaction. In this case, 2ul (approx. 5x105cpm approx. 0.5ng) of radiolabeled primer and 2ul of water can be used to replace the unlabeled primer and [32P ]dATP in the reaction (step 4), and the other steps are the same. The transfer of [ γ-32P ] of 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 articulating heads for 0.5 ml microtubes, as well as dump-flat heads and microtitre plate racks (e.g., Sorvall products) with polystyrene foam or rubber liners.
Specialized equipment
Microcentrifuge tubes (0.5 ml) or microtitre plates (flexible, heat-resistant, U-bottom 96-well wrenches with a capacity of 300ul per well).
See information section "Microtitre plates'
Methods
1. Add to the wells of a 0.5 ml microcentrifuge tube or microtitre plate:
Single-stranded template DNA (0.1ug/ul) 5ul
Oligonucleotide primer (1ug/ml, ~160pmol/m]) 4ul
TM buffer 1ul
2. Close the top of the tube or seal the microtitre plate and incubate the reaction mixture at 55°C for 5-10 min to anneal the oligonucleotide primer from the DNA template. If necessary, the annealed template and primers can be stored at -20°C for several months.
3. Label four microcentrifuge tubes or four contiguous wells of a 96-well U-titre plate with the letters C, T, A, and G; then add 4ul of the appropriate ddNTP extension/termination mixture to each tube (e.g., 4ul of ddCTP to the wells of a C-labeled microcentrifuge tube or microtitre plate, 4ul of ddTTP to the wells of a T-labeled microcentrifuge tube or microtitre plate, etc.). ddTTP mix, etc.).
4. Place the annealed primer template solution on ice and add:
[α-32P]dATP or [α-33P]dATP or [α-35S]dATP 1ul
Klenow enzyme (approx. 2.5 units) 1ul
0~1 mmol/LdATP (if [α-32P] or [α-33P] is used) 1ul
or
Water (if [α-35S]dATP is used) 1ul
Klenow enzymes should be stored without bringing them to room temperature. The enzyme will lose activity in an ice bucket for a few hours.
5. Take 3ul of the mixture from Step 4 and add it to the wall of each C, T, A, and G tube or to the side of the microtitre plate well. Do not allow the radiolabeling mix to come into contact with the ddNTP Extension/Termination Mix. The added liquid should hang on the wall of the tube or well near the edge.
6. Place the small centrifuge tubes in a microcentrifuge (with a suitable swivel 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 the tubes or plates at 2000 r/min for a few seconds to mix the reactants. Start extension/termination of the reaction. incubate at 37°C for 10-12 min.
Klenow's enzyme catalyzes the extension/termination reaction and the tracer reaction well over the temperature range of room temperature to 37°C. The enzyme can be used in a wide variety of applications.
7. After incubation, add 1 ul of tracer solution along the wall of each C, T, A, G tube or well. After a total of 10~12 min incubation, centrifuge for 2s to mix the tracer solution into the extension/termination reaction solution. Incubate at room temperature for another 10-12 min.
8. After a second incubation period of 9 to 11 min, add 6 ul of formamide sampling buffer along the wall of each C, T, A, G tube or well. Do not allow the solution to slip into the polymerization solution. At the end of the incubation, terminate the sequencing reaction by centrifugation of a microcentrifuge tube or microtitre plate.
9. The reaction can be stored at -20°C for up to 5 days and can also be analyzed directly by variable 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 each well of the sequencing gel.