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 research, the most commonly used probe is a double-stranded DNA probe, which plays a critical role in the identification and clinical diagnosis of transgenic plants. Two main methods are employed for the synthesis of double-stranded DNA probes: nick translation and random primer synthesis. These techniques are essential for creating labeled DNA fragments that can be used in hybridization experiments.

Nick translation is a widely used method for labeling DNA. It involves creating a single-strand nick on a double-stranded DNA molecule. E. coli DNA polymerase I then binds to the 3' hydroxyl end of the nick and incorporates nucleotides. At the same time, the enzyme's 5' to 3' exonuclease activity removes nucleotides from the 5' side of the nick. This process allows the nick to move along the DNA strand, replacing non-radioactive nucleotides with radioactive ones such as [α-32P]dNTPs. The resulting fragment typically ranges from 50 to 500 nucleotides in length. Several factors influence the outcome of the nick translation reaction:

• The specific activity of the final product depends on the specific activity of the [α-32P]dNTP and the extent of nucleotide replacement in the template.
• The amount of DNase and the quantity of E. coli DNA polymerase I affect the size of the resulting fragment.
• Impurities like agarose in the DNA template may inhibit enzyme activity, so it's crucial to use highly purified DNA.

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

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

(3) [α-32P]dCTP or [α-32P]dATP: 400 Ci/mM, 10 µCi/µL.

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

(5) DNase: 1 mg/mL.

(6) EDTA: 200 mM, pH 8.0.

(7) 10M NH4Ac.

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

• Unlabeled dNTP: 10 µL
• 10× Nick Translation Buffer: 5 µL
• Target DNA (1 µg): 1 µL
• [α-32P]dCTP or dATP (70 µCi): 7 µL
• E. coli DNA Polymerase I: 4 units
• DNase I: 1 µL

Add water to reach a final volume of 50 µL.

(2) Incubate the reaction mixture in a 15°C water bath 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, then add two volumes of pre-cooled anhydrous ethanol to precipitate the DNA probe.

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 ease of detection.

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