Subtractive hybridization examples

Fig. 12.1. Examples of subtractive hybridization systems. Example I shows the EcoRJ method in which the proportion of over-represented mRNA (in the + library) is increased in the final library. Simple alternatives are based on the selective removal of common sequences in — and + preparations, either by biotinylated — cDNA (II) or poly(A) tails from RNA hybridized to + cDNA (III).

A second example of subtractive hybridization took advantage of the variation of natural product production in two types of C. roseus tissue [33]. In C. roseus, roots and leaves produce a distinct spectrum of alkaloids. The leaf and root gene expression profiles were comparatively analyzed using subtractive hybridization. A total of 155 ESTs were subjected to homology-based classification and 16 EST sequences that had never been previously observed were obtained. Again, although new insights into the mechanism of the alkaloid pathway were not reported, functional analysis of these novel ESTs may provide new information into the mechanism of this alkaloid biosynthesis in C. roseus. 

In this particular example we could have avoided some of the labour involved in finding the combinations of hybrid orbitals which are equal to px and ptf, by using the 8 point group (to which the molecule also belongs). For this point group, the two-dimensional representation, the cause of all the trouble, can be expressed as two complex one-dimensionl representations. The orbitals p, and py are then just as easy to obtain as the s-orbital. Any complex numbers which result are eliminated at the end of the treatment by addition and subtraction of the orbitals formed. This is the technique which was used in 10-7 to find the 7T-molecular orbitals of the trivinylmethyl radical. It is, however, of no avail when dealing with point groups which have three-dimensional irreducible representations as in our next example, CH4. 

The examples show, that the hybrid approach clearly outperforms methods based on a single graph type. As expected, FDDs are ideally suited for bit selection from BMDs. If the size of the selected bit is small compared to the size of a BMD, this approach even outperforms BMD/ BMD subtraction, which is implicitly based on a BMD expansion. 

To create orbitals that are localized between adjacent atoms we add and subtract (also called in- and out-of-phase addition) the atomic orbitals on the adjacent atoms, which are aligned to overlap each other. For example, let s consider methane, CH (Figure 1.17). The sp hybrid orbitals point at the s hydrogen orbitals therefore, we add and subtract these atomic orbitals to create the molecular orbitals. As with H. 

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