Radiolabeling of minus cDNA probes by random oligonucleotide extension method
The synthesis of cDNA in this protocol was carried out in four saturating concentrations of dNTP and one trace of radiolabeled dNTP. After deduction of hybridization, the enriched single-stranded cDNA is labeled into a high specific activity probe by a secondary synthesis reaction of random oligonucleotide primer extension in the presence of Klenow fragments of E. coli DNA polymerase Ⅰ. This experiment is from "Guide to Molecular Cloning Experiments, Third Edition", translated by Huang Peitang et al.
Operation method
Radiolabeling of minus cDNA probes by random oligonucleotide extension method
Principle
The synthesis of cDNA in this protocol was carried out in four saturating concentrations of dNTP and one trace of radiolabeled dNTP. After subtracting hybridization, the enriched single-stranded cDNA is labeled into a high specific activity probe by a secondary synthesis reaction of random oligonucleotide primer extension in the presence of a Klenow fragment of E. coli DNA polymerase I. The cDNA is labeled by the Klenow fragment of E. coli DNA polymerase I. The dNTP concentration in the first reaction is unrestricted. Since the concentration of dNTP in the first reaction is unrestricted, the amount and size of cDNA generated is higher than in the standard protocol, and the hybridization step is therefore subtracted for high efficiency. The resulting cDNA population is less susceptible to damage by radiolysis and can be stored for long periods of time and, if necessary, labeled into higher specific activity probes.
Materials and Instruments
Escherichia coli DNA polymerase I Klenow fragment Reverse carotelase Random deoxynucleotide primers Template mRNA Move I. Materials For more product details, please visit Aladdin Scientific website.
Dithiothreitol Ethanol EDTA NaOH Placental RNA enzyme inhibitor Random primer buffer Sodium acetate dNTP solution
Sephadex G-50 columns Microtubes Water baths
1. Buffers and solutions
Dithiothreitol (DTT) (1 mol/L)
EDTA ( 0.5 mol/L, pH 8.0)
Ethanol
HCl ( 2.5 mol/L)
NaOH ( 3 mol/L)
Phenol: chloroform (1:1, V/V)
Placental RNAase inhibitor
5X random priming buffer (250 mmol/L Tris ( pH 8.0), 25 mmol/L MgCl2, 100 mmol/L NaCl, 10 mmol/L DTT, 1 mol/L HEPES (adjusted to pH 6.6 with 4 mol/L NaOH), freshly diluted aqueous solution using a 1 mol/L DTT storage solution stored at - 20°C. Diluted DTT was discarded after use.)
SDS ( 20% m/V)
Sodium acetate (3 mol/L, pH 5.2)
Tris-Cl (1 mol/L, pH 7.4)
2. Enzyme and buffer
E. coli DNA polymerase I Klenow fragment
Reverse Carboxylase
10X Reverse Transcriptase Buffer
3. Nucleic acids and oligonucleotides
(complete) dNTP solution containing 5 mmol/L of each of the 4 dNTPs
dNTP solution containing 5 mmol/L each of dCTP, dGTP, and dTTP
oligo (dT )12-18
Random deoxynucleotide primers of 6 or 7 bases in length
Template mRNA
4. Radiocomplexes
[ α-32P ] dATP ( 10 mCi/ml, specific activity >3000 Ci/mmol)
[α-32P] dCTP ( 10 mCi/ml, specific activity 800-3000 Ci/mmol)
5. Specialized equipment
Sephadex G-50 centrifuge column equilibrated with TE (pH 7.6)
Siliconized microcentrifuge tubes (1.5 ml)
Water baths preheated to 45°C, 60°C and 68°C
II. Methods
1. Mix the following components in a sterilized microcentrifuge tube at 4°C to synthesize the first strand of cDNA:
Template RNA (1 mg/ml) 10 μl
oligo(dT )12-18 (1 mg/ml) 10 μl
5 mmol/L dNTP (complete) solution 10 μl
50 mmol/L DTT 1 μl
10X reverse transcription buffer 5 μl
10 mCi/ml [α-32P] dCTP 5 μl
( Specific activity 800 or 3000 Ci/mmol)
Placental RNAase inhibitor 25 units
Water without RNAase to 46 μl
Reverse transcriptase (~800 units) 4 μl
2. Detect the percentage of radiolabeled dNTP incorporated into TCA precipitates or bound to DE-81 membranes. Calculate the yield of the first strand of cDNA using the following formula. 
3. Add the following reagents to terminate the reaction:
0.5 mol/L EDTA (pH 8.0) 2 μl
20% (m/V) SDS 2 μl
Mix the components in the microtube well.
4. Add 5 μl of 3 mol/L NaOH into the reaction tube and incubate the reaction at 68℃ for 30 min to hydrolyze the RNA.
5. Cool the reaction to room temperature. Add 10 μl of 1 mol/L Tris-Cl (pH 7.4), mix well to neutralize the solution, and then add 5 μl of 2.5 mol/L HCl. Spot a small drop of the solution (<1 μl) on pH paper to check the pH of the solution.
6. Purify the cDNA by phenol:chloroform extraction.
7. Separate the radiolabeled probe from the unadulterated dNTP by chromatography on a Sephadex G-50 centrifugal column.
8. Two rounds of subtractive hybridization are performed.
9. Concentrate cDNA by sequential extraction with isobutanol and remove salt by Sephadex G-50 chromatography.
10. cDNA is recovered by standard ethanol precipitation. cDNA is dissolved in water to a final concentration of 15 ng/μl.
11. To radiolabel the deduced cDNA with high specific activity, mix the following components in a 0.5 ml microcentrifuge tube:
Minus cDNA 5 μl
Random deoxynucleotide primer (125 μg/ml) 5 μl
12. Heat the mixture to 60°C for 5 min and then cool to 4°C.
13. Add to primer:cDNA template mixture:
5X Random Primer Buffer 10 μl
containing dCTP, dGTP and dTTP.
5 μl of dNTP solution containing 5 mmol/L each of dCTP, dGTP, and dTTP
10 mCi/ml [ α-32P ] dATP
(specific activity >3000 Ci/mmol) 25 μl
Klenow fragment (12.5 units) 2.5 μl
Water Add to 50 μl
React for 4-6 h at room temperature.
14. Add the following reagents to terminate the reaction:
0.5 mol/L EDTA ( pH 8.0) 1 μl
20% (m/V) SDS 2.5 μl
15. Separate the radiolabeled cDNA from the undoped dNTP by chromatography on a Sephadex G-50 centrifugal column.