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 is widely used in the identification and clinical diagnosis of transgenic plants. There are two main methods for synthesizing double-stranded DNA probes: nick translation and random primer synthesis.

Nick translation When a nick is made on one strand of a double-stranded DNA molecule, E. coli DNA polymerase I binds the nucleotide to the 3' hydroxy terminus of the nick. At the same time, the enzyme has an exonuclease activity from 5' to 3', which can remove nucleotides from the 5' end of the nick. Since the nucleotide is added to the 3' end of the nick while the nucleotide is removed, the nick is moved along the DNA strand, and the radioactive isotope is replaced by a radioactive nucleotide instead of the originally non-radioactive nucleotide. To the synthesis of a new chain. The most suitable nick translation fragment is typically 50-500 nucleotides. The nick translation reaction is affected by several factors: (a) The specific activity of the product depends on the specific activity of the [α-32P]dNTP and the extent to which the nucleotides in the template are replaced. (b) The amount of DNase and the mass of E. coli DNA polymerase I affect the size of the product fragment. (c) Inhibitors such as agarose in the DNA template inhibit the activity of the enzyme, so carefully purified DNA should be used.

Experimental reagent

(1) 10× nick translation buffer: 0.5 M/L Tris-Cl (pH 7.2); 0.1 M/L MgSO4; 10 mM/L DTT; 100 ug/ml BSA.

(2) Unlabeled dNTP stock solution: Except for the isotope-labeled deoxynucleotide nucleotides, the other three were dissolved in a 50 mM/L Tris·Cl (pH 7.5) solution at a concentration of 0.3 mM/L.

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

(4) E. coli DNA polymerase I: (4 units/ul): dissolved in 50 ug/ml BSA, 1 mM/L DTT, 50% glycerol, 50 mM/L Tris·Cl (pH 7.5).

(5) DNase: 1 mg/ml.

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

(7) 10M/L NH4Ac.

Experimental procedure

(1) Mix according to the following ratios:

Unlabeled dNTP 10ul

10× nick translation buffer 5ul

DNA to be labeled 1ug

[α-32 P]dCTP or dATP (70uCi) 7ul

E.coli DNA polymerase 4 units

DAN enzyme I 1ul

Add water to a final volume of 50ul

(2) Place in a 15 ° C water bath for 60 minutes.

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

(4) Ammonium acetate was added to the reaction solution to a final concentration of 0.5 M/L, and a DNA probe was recovered by adding two volumes of pre-cooled anhydrous ethanol to precipitate.

Precautions

1, 3H, 32P and 35S labeled dNTPs can be used for probe labeling, but [α-32P]-dNTP is commonly used.

2. The activity of DNase I is different, and the specific activity of the obtained probe is also different. When the activity of DNase I is high, the specific activity of the obtained probe is high, but the length is relatively short.