Experimental process of nucleic acid probe labeling - Database & Sql Blog Articles

Probe domestic double-head probe 038-BB-5.7L The shape of the two ends of the needle is outside the pointed needle

In molecular biology, the most commonly used probe is a double-stranded DNA probe, which plays a crucial role in identifying and diagnosing transgenic plants. Two primary methods are employed to synthesize these probes: nick translation and random primer synthesis.

Nick Translation: This technique involves creating a single-strand break (nick) on one strand of a double-stranded DNA molecule. E. coli DNA polymerase I then binds to the 3' hydroxyl end of the nick and adds nucleotides, while its 5' to 3' exonuclease activity removes nucleotides from the 5' side. This process results in the movement of the nick along the DNA, replacing non-radioactive nucleotides with radioactive ones such as [α-32P]dNTPs. The typical fragment size produced by this method ranges between 50 and 500 nucleotides.

The success of the nick translation reaction depends on several factors. First, the specific activity of the product is influenced by the quality of the [α-32P]dNTPs and the extent of nucleotide replacement in the template. Second, the amount of DNase and the concentration of E. coli DNA polymerase I affect the size of the resulting fragments. Lastly, inhibitors like agarose in the DNA template can interfere with enzyme activity, so it's essential to use highly purified DNA for optimal results.

(1) 10× Nick Translation Buffer: Contains 0.5 M Tris-Cl (pH 7.2), 0.1 M MgSO4, 10 mM DTT, and 100 µg/ml BSA.

(2) Unlabeled dNTP Stock Solution: Composed of three unlabeled deoxynucleotides dissolved in 50 mM Tris·Cl (pH 7.5) at a concentration of 0.3 mM.

(3) [α-32P]dCTP or [α-32P]dATP: Available at 400 Ci/mM with a concentration of 10 µCi/µL.

(4) E. coli DNA Polymerase I: Supplied at 4 units/µL, dissolved in 50 µg/ml BSA, 1 mM DTT, 50% glycerol, and 50 mM Tris·Cl (pH 7.5).

(5) DNase I: Concentration of 1 mg/ml.

(6) EDTA: 200 mM (pH 8.0).

(7) 10 M Ammonium Acetate.

(1) Prepare the reaction mixture using the following components:

• Unlabeled dNTP: 10 µL
• 10× Nick Translation Buffer: 5 µL
• DNA to be labeled: 1 µg
• [α-32P]dCTP or dATP (70 µCi): 7 µL
• E. coli DNA Polymerase I: 4 units
• DNase I: 1 µL
• Add water to bring the total volume to 50 µL.

(2) Incubate the mixture at 15°C for 60 minutes.

(3) Stop the reaction by adding 5 µL of EDTA.

(4) Add ammonium acetate to achieve a final concentration of 0.5 M and precipitate the DNA probe by adding two volumes of pre-cooled anhydrous ethanol.

1. While 3H, 32P, and 35S-labeled dNTPs can all be used for probe labeling, [α-32P]-dNTP is the most commonly used due to its high sensitivity and stability.

2. The activity of DNase I varies, which directly affects the specific activity and length of the resulting probe. A higher DNase I activity leads to a higher specific activity but shorter fragment lengths.