Protocol:Reverse dot blot (RDB)

Reverse dot blot (RDB)
Reverse dot-blot analysis is a technique for immobilizing allele-specific oligonucleotide probes on a nylon membrane rather than the individual DNA samples. This is a non-radioactive method. In this format, multiple pairs of mutant and normal ASO probes are spotted on strips of nylon membranes. For each diagnostic test, a spotted strip containing many normal and mutant oligonucleotides, is hybridized with a specific DNA probe to screen many mutations. Reverse dot-blot analysis was first described by Saiki et al (22), and then developed later to screen many β-thalassemia mutations in Sicilian population and for use in prenatal diagnosis (23,24).

The reverse dot-blot can detect most common and uncommon specific mutations present in a determined country after the conditions are carefully set-up. No single RDB can cover all the mutations in any country. RDB is also used in Sicily to analyse &alpha;-thalassaemia (25) and &delta;-thalassaemia point mutations, and for the genotyping of the main &beta;-globin gene variants (HbS, HbC, HbD, etc). A general outline of the method is described.

Method

 * 1) The amplified DNA is labelled using 5’ modified primers with biotine or during the amplification by biotine-16-dUTP.
 * 2) Oligonucleotide probes are 5' amino-modified. A NH2 group is added during the last step of the synthesis.
 * 3) Membrane (Biodyne-C, PALL-Biosupport) is activated by EDC and the oligonucleotide probe is spotted on the surface of the membrane using a 2 &mu;L pipette. On the left and on the right of the same lane are spotted the normal and the mutant oligonucleotides.
 * 4) After inactivation of membrane, it is pre-hybridized for 15 min at 45oC.
 * 5) Amplified DNA is diluted in hybridization solution and denaturated at 95oC for 10 minutes. Then it is added to the pre-hybridised membrane.
 * 6) Membranes are incubated for 60 min. at 45oC and then washed for 20 min at 45oC.
 * 7) Conjugation step with streptavidin-AP is performed for 30 min at room temperature.
 * 8) The membranes are washed for 15 min at room temperature.
 * 9) Finally, the filters are incubated in a solution containing NBT/BCIP and the color is developed in 30-45 min.

A critical requirement of this assay is the optimization of single washing temperature for all probes. This requirement is best controlled by optimizing the length and amount of each oligonucleotide probe. This usually requires the synthesis and trial of many probes. In some instance, the addition or the deletion of a single nucleotide from the sequence of the oligonucleotide probe is critical for correct typing. The membrane strips containing the covalently bound oligonucleotide probes can be prepared in large batches and stored ready for use at room temperature for six months.

Interpretation of results
It is important to emphasise that there must be a differentiation between false positive due to background signals and the true positive results of the control samples. If in doubt the assay should always be repeated. The strategy, as with ARMS-PCR, is to screen for the common mutations on one membrane and then if no positive result is obtained, the rare ones are screened for on a second membrane. Any mutation remaining unidentified is then investigated by direct sequencing.

Figure 5.2 shows illustrates the main steps involved in the generation of a Reverse Dot Blot using immobilized ASO probe.

Image

Figure 5.3 shows RDB being used to screen for the 7 most common mutations found in Sicily (as listed in Table 5.3). These account for 95% of the &beta;-thalassaemia defects in Sicily.

Image

Figure 5.4 shows RDB being used for prenatal diagnosis of &beta;-thalassaemia in Dr. Maggio’s laboratory in Sicily.

Image

Figures 5.5 and 5.6 show RDB being used to detect &alpha;-thalassaemia and &delta;- thalassaemia point mutations in Dr. Maggio’s laboratory.

Image Image