Protocol:Allele specific oligonucleotide (ASO) dot blot

Allele specific oligonucleotide (ASO) dot blot
Dot blot analysis is a technique for immobilizing amplified DNA on a nitrocellulose or nylon membrane. Hybridization analysis can be carried out to determinate the relative target sequence in the blotted DNA preparations. Dot blot analysis was first described by Kafatos et al (21). This procedure was originally used to determinate the relative amount of a target sequences in a series of DNA samples. A large number of samples can be applied simultaneously on the surface of the membrane, enabling many different DNAs to be screened in a single hybridization experiment, or in sequential hybridisation experiments to investigate several different mutations.

Reagents

 * 1) 20x SSPE
 * 2 M NaCl
 * 0.2 M NaH2PO4.2H2O
 * 0.02 M Na2EDTA, pH 7.4 (with 10 M NaOH)
 * 1) 50x Denhardt’s
 * 1% Ficoll
 * 1% Polyvinylpyrrolidene (PVP)
 * 1% Bovine serum albumin (BSA)

Method
Samples are usually applied to the membrane using a manifold attached to a suction device especially if a number of blots are to be prepared at any time. Samples of amplified DNA to be transferred are denaturated (by adding 10x volume of 0.4 M NaOH, 25 mM Na2EDTA) or heat (80 oC for 5 min) and applied to the membrane in a salt buffer. After blotting, the membrane is treated (washed) with denaturant (0.4 M NaOH) and neutralization solutions (2x SSC) and the DNA immobilized by incubation in vacuum at 80 oC (nitrocellulose membrane) or UV irradiation (nylon membrane).

Once the membrane has been prepared with the immobilized denatured amplified DNA, the hybridisation steps can follow. Oligoprobes (ASO’s) for each mutation under investigation, (and their wild-sequence equivalent) are usually designed to be 19bp long, with the expected mutated nucleotide exactly in the middle. The ASO is labelled before hybridisation. One protocol is to label the ASO at the 5' end with &gamma;32ATP as follows: 250 ng of oligoprobe, 125 &mu;Ci of radioactivity, 10 units of T4 Kinase and 10x T4 Kinase buffer in a final volume of 20 &mu;l, and incubation at 37 oC for 2 hours. After the reaction 1 &mu;l of 0.5 M EDTA and 480 &mu;l of H2O are added. The unicorporated radioactive dNTP is removed by passing the reaction through a NAP-5 column:


 * 1) Prepare the column by passing through 3 volumes of H2O.
 * 2) Load the labelled ASO (500 &mu;l) on to the column and allow it to run in, discarding the initial eluent.
 * 3) Wash the column with 1 ml H2O and collect the radioactive eluent in a 1.5 ml eppendorf tube (count if required).
 * 4) For hybridisation, add the 1.5 ml of labelled probe to 8.5 ml of hybridisation buffer (5x SSPE, 0.5% SDS and 5x Denhardt’s). (Following the hybridisation step the 10 ml of radioactive probe in hybridisation buffer can be frozen at –20oC for up to 4-6 weeks, or as long as the radioactivity gives a signal).

The hybridisation step is performed for 2 hours at a temperature around 2oC less than the Tm of the probe (where the Tm is calculated by the simplified formula: Tm = 4x [G+C] + 2x [A+T]). After a suitable incubation, the membrane is washed twice at room temperature (10x SSPE and 0.5% SDS) for 15 minutes and then once at the Tm of the ASO (4x SSPE and 0.1% SDS). Any non-specifically bound probe is removed, leaving only probe that is base-paired to the target DNA.

After hybridization and wash, the membrane is subjected to autoradiography for 12- 16 hours. The membrane can be stripped (by treating with 0.4 M NaOH at 42oC for 30 minutes, followed by neatralizing with 0.2 M Tris/HCl, 0.2x SSC and 0.5% SDS) and then re-hybridized with another probe. This method needs one analysis for one mutation. For this reason, you need a lot of the time to screen many mutations. Moreover, the temperature of hybridization and wash for each oligonucleotide is different. In the same procedure it is possible to study many samples for one mutation.

Figure 5.1 shows an example of dot blot analysis (isotopic).

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