R-loop mapping is a technique in which mRNA is hybridized with double stranded DNA under conditions in which RNA-DNA hybrid is more stable than the DNA duplex. This allows displacement of one strand of DNA in the region, where it can pair with RNA. When DNA segment belonging to an interrupted gene is used in such an experiment for hybridization with mRNA, the intervening sequence or intron region loops out as double stranded DNA and the exon region loops out as single stranded DNA loops as shown in Figure 29.3. The double stranded DNA loop will appear at the junction of two single stranded loops produced due to displacement by mRNA. These loops can be observed under the electron microscope as has been done for mouse minor β-globin gene showing single intron. This technique is comparable to the hybridization of mRNA with single stranded DNA described earlier. An interrupted gene can also be studied by comparing the restriction map of the interrupted gene with that of its corresponding cDNA prepared from its mRNA through reverse transcription. When this is done, there may be two situations : (i) If the intervening sequence or intron region does not have a recognition site for an enzyme, there is only a change in the length of corresponding segment (since the two restriction sites in the regions on either side of an intron are now separated by a longer distance) but the number of segments remains the same (Fig. 29.4). (ii) If the intron region has a restriction site for the enzyme employed, an extra cut will be made in this region. This extra cut will produce two fragments corresponding to one segment in the cDNA (Fig. 29.5).
Fig. 29.3. R-looping, where mRNA is hybridized with double stranded DNA (note the double stranded thick loop showing intron region and single stranded thin loops representing the exons).
Fig. 29.4. Restriction fragment analysis of a part of interrupted gene and its cDNA, showing that the length of a segment A (which had no restriction site) is decreased in cDNA due to splicing out of intron region.
Fig. 29.5. Restriction fragment analyses of a part of interrupted gene (having a restriction site in the intron region A, dividing it into two regions A1 and A2) and its cDNA showing decrease in the number of fragments in cDNA from four to three.
Fig. 29.6. A comparison of the restriction maps of cDNA and genomic DNA for mouse p-globin gene, showing that the gene has two introns not present in cDNA. Only one of them shows up as a loop during hybridization of mRNA with denatured single stranded DNA (since the second intron is very small).
When restriction maps are prepared for cDNA and the gene using a number of enzymes, we may find that the two maps may be similar at the two ends but extra sites may be present in the intron regions of the gene, which are absent in cDNA or mRNA. The resolving power of restriction mapping allows detection of gene segments upto 20-30 bp long. Restriction maps of cDNA and genomic DNA of mouse β-globin gene are shown in Figure 29.6. It may be seen that there are two intron regions in the gene; one is much larger, which can be visualized in R-loops under the electron microscope, but the other is smaller, that can not be visualized under the electron microscope. This demonstrates the higher resolving power of restriction mapping.