Oligonucleotide 5' terminal phosphorylation assay
The following describes a reaction for labeling a 10 pmol high specific activity oligonucleotide. Labeling of different amounts of oligonucleotides can be accomplished by increasing or decreasing the volume of the reaction while maintaining the appropriate concentration of each component. Similar reaction conditions can also be used to add non-radioactive phosphoric acid to the 5' end of synthetic oligonucleotides used for targeted mutagenesis. This experiment was derived from Molecular Cloning Laboratory Guide (3rd Edition), Previous Edition, by Peitang Huang.
Operation method
Oligonucleotide 5' end phosphorylation assay
Materials and Instruments
T4 phage polynucleotide kinase buffer Tris-Cl T4 phage polynucleotide kinase oligonucleotide [γ-32P]ATP Move makings For more product details, please visit Aladdin Scientific website.
Microcentrifuge tubes Water bath
Buffers & Solutions
Dilute the storage solution to the appropriate concentration
10XT4 Phage Polynucleotide Kinase Buffer
Tris-Cl(1mol/L,pH8.0)
Enzyme and Buffer
T4 phage polynucleotide kinase
Wild-type T4 phage polynucleotide kinase is a 5' phosphotransferase and a 3'-phosphatase (Depew and Cozarelli 1974; Sirotkin et al, 1978). In contrast, the mutant enzyme (see Cameron et al. 1978) has lost phosphatase activity and retains phosphotransferase activity completely and is commercially available (Behringer Mannheim). Therefore, if possible, we recommend using the mutant enzyme for 5' end labeling. 10-20 units of enzyme are required to catalyze the phosphorylation of 10-50 pmol of 5' end phosphate-free ends.
IMPORTANT: The activity of polynucleotide kinases available from different vendors to catalyze the 5' end phosphorylation of single-stranded synthetic oligosiderophores varies widely (van Houten et al. 1998)
Nucleic acids and oligonucleotides
Oligonucleotides
To achieve the best labeling results, oligonucleotides were purified by reversed-phase chromatography as provided in Scheme 1. The labeling efficiency of polynucleotide kinase-catalyzed crude oligonucleotides is very low (vanHoutenetftL1998), and if unpurified oligonucleotides are used, it must be ensured that the final round of the synthesis cycle is set up with a triphenylmethyl closure procedure, i.e., that the dimethoxydiphenylmethyl closure group of the oligonucleotide primer must be removed prior to the oligonucleotide's dissociation from the solid phase carrier. The dimethoxytrityl closure group effectively prevents modification of the hydroxyl group at the 5' end of the oligonucleotide.
Radioactive compounds
[ γ-32P ]ATP (10 mCi/ml, specific activity >5000 Ci/mmol) solution
10 pmol of [ >32P ]ATP is required to label the 5' end of the phosphate-free terminus at a high specific activity of 10 pmol. In order to minimize the radiolysis of the precursor compound and the probe, [32P ]ATP should be labeled on the same day as the arrival of [32P ]ATP as far as possible.
Specialized Equipment
Microcentrifuge tube (0.5 ml)
Water bath pre-warmed to 68°C
Additional reagents
The reagents required for step 4 of this protocol are listed in Protocol 7 of Chapter 13 or in Protocol 5 of this chapter.
Methods
1. Prepare the reaction mixture in a 0.5 ml microcentrifuge tube.
Synthetic oligonucleotide (10 pmol/ul) 1ul
10xT4 phage polynucleotidase buffer 2ul
[ γ-32P ]ATP (10pmol, specific activity >5000Ci/mmol) solution 5ul
Water 11.4ul
Keep flicking the walls of the centrifuge tube to mix the reaction mixture well, take 0.5ul of the reaction mixture and add it to a centrifuge tube containing l0ul of 10 mmol/L Tris-Cl (pH 8.0) to be used in step 4.
The reaction solution contains equal concentrations of [ γ-32P ]ATP and oligonucleotides. Typically only 50% of the radiolabel is transferred to the oligonucleotide, and a 10-fold increase in oligonucleotide concentration increases the transfer efficiency by up to 90%. However, the specific activity of the radiolabeled oligonucleotide is also reduced by a factor of about 5. To label the highest specific activity oligonucleotides, the following measures can be used:
-Increase the concentration of [ γ-32P ]ATP in the reaction solution by a factor of 3 (i.e., use 15ul of radiolabel and reduce the volume of water to 1.4ul).
-Decrease the amount of oligonucleotide to 3 pmol.
Under these conditions, only about 10% of the radiolabel was transferred to the oligonucleotides, but a high percentage of oligonucleotides were radiolabeled.
Ideally, a 5-fold molar excess of ATF should be present at the end of the DNA at a concentration greater than 0.4 pmol/L, and the resulting concentration of ATP in the reaction solution should be greater than 2 umol/L, but this is difficult to achieve in practice.
2. Add 10 units (~1ul) of T4 phage polynucleotide kinase to the remaining reaction solution, mix well and incubate at 37°C for 1h.
3. At the end of the incubation, 0.5ul of the reaction solution is removed and transferred to a second centrifuge tube containing 10ul of 10 mmol/L Tris-Cl (pH 8.0). The remaining reaction solution was heated at 68°C for 10 min to inactivate the polynucleotide kinase. The heated reaction solution was kept on ice.
4. Before proceeding to the next step, test a small portion of the removed labeled reaction solution to determine if the labeling reaction is effective by measuring the transfer of the radiolabel from the oligonucleotide substrate. Take a sample (0.5ul) and add it to a new centrifuge tube containing 10ul of 10mmol/L Tris-Cl (pH 8.0). Combine this sample with the samples taken in Steps 1 and 3 of the assay and test the transfer efficiency of γ-32P by one of the following methods.
-Determine the proportion of radioactivity bound to the DE-81 filter membrane. Oligonucleotides bind strongly to this positively charged membrane, while [ γ-32P ]ATP does not. Details of the method can be found in Scheme 7 in Chapter 13.
or
-Estimation of the radiolabeled fraction migrating with the oligonucleotide and determination of the efficiency of the labeling reaction by size exclusion chromatography with a G-15 or Bio-Rad P-60 column. This method is simpler in comparison because the amount of incorporated and unincorporated radioactivity can be detected on the chromatography column with a hand-held microprobe.
5. If the specific activity of the oligonucleotide is acceptable, the radiolabeled oligonucleotide can be purified using Schemes 3, 4, 5, and 6.
If the specific activity is too low, add 8 additional units of polynucleotide kinase and continue incubation at 37°C for 30 min (90 min total). Inactivate the polynucleotide kinase by heating at 68°C for 10 min, and analyze the reaction product as described in step 4.
If the oligonucleotide produced by an additional round of scaling does not meet the specific activity requirements of the current experiment, check to see if the oligonucleotide contains cytosine at the 5' end, and if so, consider redesigning the oligonucleotide so that it has an A, T, or G at the 5' end (see Overview of this protocol). Alternatively, try purifying the initial oligonucleotide by Sep-Pak chromatography and then repeat this experiment.