The efficiency of nucleotide incorporation in DNA/RNA polymerization reactions (e.g. transcription, reverse transcription, and DNA replication) can be determined by trichloroacetic acid (TCA) precipitation. TCA precipitates nucleic acid polymers longer than ~20 nucleotides and can therefore be used to separate radiolabeled nucleotides incorporated into nucleic acid from unincorporated label.
The following is a protocol for monitoring the efficiency of radiolabeled nucleotide incorporation in a polymerization reaction by TCA precipitation. In the protocol, nucleic acid synthesis reactions are carried out in the presence of a radiolabeled nucleotide (e.g., 32P-dATP or 32P-UTP). Then, the synthesized polymers are TCA precipitated and reaction efficiency is determined by the following formula:
cpm per Ál of sample after TCA precipitation
cpm per Ál of sample with no precipitation
The protocol has been optimized with materials from the listed vendors. The specific details (e.g. size of tubes, amounts of carrier and sample) are arbitrary and can be varied according to user preference.
Reagents and Equipment Required
- Borosilicate tubes, 12 x 75 mm (e.g., Fisher Cat #14-961-26)
- 10% TCA (e.g., Acros Cat # 42145-5000; make a 10% (w/v) solution in water) Warning: TCA is caustic!
- Carrier nucleic acid, 1 mg/ml (e.g., sheared fish sperm DNA, Ambion Cat #9680; dilute to 1 mg/ml in water)
- Glass fiber filters to catch precipitate (e.g., Whatman GF/C filter circle; or Schleicher & Schuell #34 2.7-cm glass filter circles, Cat #10373828)
- Aqueous scintillation fluid (e.g., EcoLume, ICN Cat #882470)
- Vacuum manifold (e.g., Millipore Cat #XX270550)
- Scintillation vials, Scintillation counter, vortexer, pipettors
- Dispense 198 Ál of carrier DNA (1 mg/ml) into a 12 X 75 mm glass tube.
- Add 2 Ál of the nucleic acid synthesis reaction and mix thoroughly.
- Transfer 100 Ál of the diluted DNA/RNA synthesis reaction to aqueous scintillation cocktail and count in a scintillation counter. The counts per minute (cpm) will reflect the total amount of radiolabel present in the reaction mixture (both unincorporated and incorporated counts).
- Add 2 ml of cold 10% TCA (trichloroacetic acid) to the 12 X 75 mm tube containing the remaining 100 µl diluted DNA/RNA. Mix thoroughly and place on ice for 10 minutes. This will precipitate nucleic acids longer than ~20 nucleotides.
- Collect the precipitate via vacuum filtration through a Whatmann GF/C glass fiber filter (or equivalent). Prewet the filter with a small amount of 10% TCA prior to adding the sample.
- Rinse the tube twice with 1 ml of 10% TCA and then rinse once with 3-5 ml of 95% ethanol. Pass each of the rinses through the GF/C filter.
- Place the filter in a scintillation vial, add aqueous scintillation cocktail, and count in a scintillation counter. The cpm will reflect the amount of radiolabel that was incorporated.
Calculate the Fraction of Label Incorporated:
- Divide the cpm of the TCA precipitated sample (step 7) by the cpm of the non-TCA precipitated sample (step 3) to determine the fraction of label incorporated (multiply by 100 for percent incorporation).
For help in determining the specific activity, theoretical yield and actual yield of your reaction products, please see the following online calculators:
RNA Probe Specific Activity Calculator
DNA Probe Specific Activity Calculator