Experiments with oligonucleotides synthesized by labeling with Escherichia coli Klenow fragments
Experiments with oligonucleotides synthesized by labeling with Escherichia coli Klenow fragments
Oligonucleotides are usually labeled using a phosphorylation reaction catalyzed by the T4 phage polynucleotide kinase. However, there is sometimes a need to label radiolabeled probes with higher activity than is desirable, ideally with one 32P atom doped on each oligonucleotide molecule in the phosphorylation reaction. In contrast, synthesis of a DNA strand complementary to the oligonucleotide using the IKlenow fragment of E. coli DNA polymerase results in a higher specific activity probe. This experiment is from the first volume of Laboratory Guide to Molecular Cloning (3rd edition), by Peitang Huang.
Operation method
Experiments with oligonucleotides synthesized by labeling with Escherichia coli Klenow fragments
Materials and Instruments
Formamide spiking buffer E. coli DNA polymerase IKlenow fragments Denaturing polyacrylamide gels Oligonucleotide primers Oligonucleotide templates [α-32P]dNTP Move makings For more product details, please visit Aladdin Scientific website.
Phosphofluorescent paste labels Water bath or module
Solutions and buffers
Dilute the sample storage solution to the appropriate concentration.
Formamide spiking buffer
10X Klenow Buffer
Enzyme & Buffer
E. coli DNA Polymerase IKlenow Fragments
Gel
Denaturing Polyacrylamide Gel
The proportion of acrylamide in the gel solution and the electrophoretic conditions vary depending on the size of the oligonucleotides in the reaction mixture, and Table 10-4 lists some useful hints: 
Polyacrylamide gels are usually filled with lxTBE (89 mmol/LTris-borate, 2 mmol/LEDTA). Electrophoresis is also performed in this buffer. The method and procedure for filling polyacrylamide gels are described in Scheme 1 and Scheme 8 in Chapter 12.
Nucleic acids and oligonucleotides
Oligonucleotide Primers
Purify primers as described in Scheme 1. To ensure efficient labeling, the primer concentration in the reaction solution should be 3 to 10 times molar excess in the DNA template.
Oligonucleotide template
Oligonucleotide templates are purified using the method described in Scheme 1. The sequence of the oligonucleotide template should be complementary to the sequence of the desired radiolabeled probe.
Radioactive compounds
[ α-32P ]dNTP
In order to keep the concentration of the reaction substrate high, the chain extension reaction should be carried out in the smallest possible volume. Therefore, it is preferable to use radiolabeled dNTP dissolved in ethanol-water rather than in buffered aqueous solutions. An appropriate volume of alcohol-soluble [ α-32P ]dNTP can be mixed and evaporated to dryness in the microcentrifuge tube to be used for the labeling reaction. To minimize radiolysis of precursor compounds and probes, the probes should be radiolabeled on the same day that the [32P ]dNTP arrives, if possible.
Specialized Equipment
Phosphor-fluorescent paste labels (commercially available) or very hot grade radioactive ink paste labels Radioactive ink is made by mixing a small amount of 32P with waterproof black drawing ink. The ink used in this program is "extreme heat grade" (hand-held compact detector counts greater than 2000 cps), and the ink is applied to the stick-on label with a fiber-tip pen. The fiber-tip pen should be labeled with a special marking tape for radioactive materials and stored in a safe place. Chemiluminescent labels are an alternative to radioactive ink and are available from Stratagene (Glogos).
These labels can be reused several times, but must be fluorescently irradiated before the next radiographic autoradiography.
Pre-warm to 80°C in a water bath or module.
Additional Reagents
The reagents required for Step 9 of this protocol are listed in Chapter 5, Protocol 12.
Methods
1. Calculate the amount of [ α-32P ]dNTP needed to achieve the specific activity requirement and sufficient for complete synthesis of the entire template chain by adding it to a microcentrifuge tube (see overview of this protocol).
The concentration of dNTP should not fall below 1 umol/L at any stage of the reaction. To maintain a high concentration of the reaction substrate, the tensile reaction should be carried out in as small a volume as possible.
2. Add the appropriate amount of oligonucleotide primer and template oligonucleotide to the centrifuge tube.
To ensure effective radiolabeling, the concentration of primer in the reaction solution should be 3-10 times molar excess of template DNA.
3. Add 0.1 volume of 10X Klenow Buffer and mix well.
4. Add Klenow fragments at 2-4 units per 5ul of reaction volume and mix well. incubate the reaction for 2-3 h at 14°C.
If necessary, the progress of the reaction can be monitored by removing small (0.1ul) samples during the reaction and determining the proportion of radioactivity precipitated by 10% trichloroacetic acid (TCA) (see Appendix 8).
5. Dilute the reaction mixture with an equal volume of formamide spiking buffer, heat at 80°C for 3 minutes, and add the entire sample to a denaturing polyacrylamide gel.
6. At the end of electrophoresis, remove the electrophoresis unit and adhere the polyacrylamide gel to the side glass (see Scheme 11 in Chapter 12 for details).
WARNING: Undoped [ α-32P ]dNTP may migrate into the lower layer of the electrophoresis sludge and make it radioactive. The gel, glass plates, buffer and electrophoresis apparatus should be treated as radioactive sources. They should be handled with care behind a protective screen of polyisobutylic acid resin.
7. Wrap the gel and the glass plate on its back together in Saran wrapping film, note the position of the indicator dye, and test the activity of the oligonucleotide-containing area on the gel with a hand-held minidetector. Apply a set of radioactive ink or phosphorescent dot labels to the wrap around the edge of the sample and cover the radioactive labels with Scotch tape to prevent radioactive ink from contaminating the film holders or the sensitizing screen.
8. Expose the gel on radioactive autoradiographic film (see Appendix 9).
The activity of the doped probe is usually so high that it takes only a few seconds to obtain a film image.
9. After the film has been developed, the image of the radioactive ink is overlaid with the radioactive label to locate the position of the probe on the gel. The strip is cut and the radiolabeled oligonucleotide is recovered as described in Scheme 12 in Chapter 5.