CATEGORIAS ARQUITECTONICAS

acceptor 6 tct Gtt ccc tgc agC 469 5 ’ y y y y yny ag 3 ’ acceptor 1 c ttc ccc ag acceptor 2 t tAA tgc ag acceptor 3 t cAt ttt ag acceptor 4 c ttA ctt ag acceptor 5 t tcc tgc ag acceptor 6 t ccc tac ag c. 493 5’nn wgg trw gt 3’ donor I at Ggg taa gt donor 2 at GTg taa gt

donor 3 ca agg tga TC

donor 4 ta tgg tga gt

donor 5 tg agg tga gt

donor 6 cc tgg tga gt

Table 3.7 Splice sites in the PLP gene and their homologies to degenerate splice site oligos a. 406, b. 469 and c. 686. Bold typed capitals indicate where mismatches with the oligo are expected.

Oligo 406 shows fiill complementarity only to acceptor 1 and should therefore only hybridize to the 6 Kb fragment from the 34H1 Hindlll/Xbal digest, the 7Kb insert of 34S1 (Sail) and

the 5.5 Kb fragment of the 34S1 Sall/BamHI digest. 406 hybridizes to these fragments and to several others. Oligo 469 should recognize acceptor 1, 5 and 6 and should therefore

hybridize to the 6Kb fragment of 34H1 (H/X), the 1Kb insert of 81H3 (H), the 7kb fragment

of 34S1 (S) and the 5.5kb fragment of 34S1 (S/B). Whilst 469 recognized these four bands as predicted, it also bound weakly to a ~ 0.8Kb 34S1 (S/B) fragment and to the vector band. Oligo 493 shows full complementarity with donors 4, 5 and 6 and so should hybridize with

34H1 (X/H) 2Kb, 81H3 (H), 34S1 (S), 34S1 (S/B) 0.4 and 0.75Kb. Whilst 493 correctly hybridized to 81H3 (H) and 34S1 (S), it also hybridized to the 6Kb 34H1 (H/X) and 5.5Kb

34S1 (S/B) fragments, but not to 34S1 (S/B) 0.4Kb and 34H1 (H/X) 2Kb.

3.4 DISCUSSION

The experiments discussed in 3.2 show that the short 8-mer oligo based on the

Not! site is able to hybridize to target DNA over a fairly wide range of temperatures and at temperatures much higher than that predicted by the formula of Suggs et al (1981). The hybridizations were also able to discriminate between correct target site, present only in pWE15 and pMBgpt and sites of 6 and 7 base pair homology present in pWE15 (the positions

of these sites are shown in Chapter 4.7) and pCV108.

Wallace et al (1979) showed that it is possible to discriminate perfect hybrids from ones containing a single internal mismatch with oligos 11-17 nucleotides long. From the evidence shown in 3.2 the same applies to 8-mer oligos with mismatches either internally or at the

ends. According to Drmanac et al (1990) shorter oligos give better discrimination for hybridization because the relative decrease in hybrid stability with a single mismatch is greater than for longer probes (T„ for a 20-mer with 1 mismatch decreases by 5-7.5“C; 1-

1.5®C per 1% of oligo mis-paired).

The experiments using degenerate splice site oligos on model genes were less successful. The oligos were unable to distinguish the correct sites (with full complementarity to known splice sites) from other sites in the clones. This could be partly blamed on the low stringency of the hybridizations and washings. However, a number of expected hybridizations were not observed, notably the 3.8Kb Bglll fragment of cosGSTrpT, which should hybridize with oligos 406, 493 and 469, and the 2Kb insert of 34H1, which should hybridize with 493. Other inconsistencies are also observed, such as the hybridization of oligo 406 to the cosGSTrp? BamHI 11.5 fragment, but not to the corresponding fragments of the other digests. If it is assumed that the restriction map of cosGStrp? is correct, then doubts over the reliability of such oligos are inevitable. Inconsistencies have since been observed in the Bglll restriction map of cosGSTrpT.

The fact that the degenerate oligo will have a wide range of Tj- depending on which bases occur at the degenerate positions in any sub-species of the oligo- could be partly responsible for this unreliability. For instance oligo 406 will have Tj between 36 and 78°C, 469 will have Tj between 2 0 and 34°C and 6 8 6 will have Tj between 30 and 38°C, according to the formula

of Suggs et al (1981). The ideal temperature for hybridization will depend on the sequence of the target splice sites, but since these sequences would be unknown in an experimental situation, the hybridization would have to be performed at an approximated average temperature. This hybridization at just one temperature may suit only a small fraction of the degenerate oligos and might encourage hybridization of other sub-species of the oligo under non-optimum conditions, thus producing background signal. However, this is insufficient to explain the lack of observation of several of the expected hybridizations. The hybridizations were performed under low stringency conditions (high salt concentration and at room

temperature) and should thus have promoted all the expected hybridizations, although rate of hybridization would probably be well below optimum at such relaxed stringency.

Many of the problems experienced using degenerate oligos could be overcome by substituting the degenerate positions in the oligos with base homologues that have ambiguous pairing abilities. For instance 5-fluorodeoxyuridine could be used to pair with A or G (Habener et al, 1988) and could be used to replace pyrimidine degeneracies (C or T). Deoxyinosine which can pair with A,T,G or C (although more so with A or C) could be used to replace either fully degenerate bases (N) or keto-bases (G or T)(Martin et al, 1985; Ohtsuka et al, 1985; Takahashi et al, 1985). Deoxyguanosine could be used at positions of two-fold degeneracy between purines G and A, because the G-T base pairing is thought to be one of the most stable mismatches (Millican et al, 1984). In this way the consensus sequences could be rationalized to allow oligonucleotides with much less base degeneracy. Such an oligonucleotide would be much easier to optimize hybridization conditions for. However, of these base homologues, only deoxyinosine is commercially available for incorporation into custom synthesized oligonucleotides.

Another type of base substitution has been shown to increase the stability of homoduplex without increasing heteroduplex stability. The incorporation of 5-bromodeoxycytosine or 5- methyldeoxycytosine in place of deoxycytosine bases in oligos has been shown to achieve this effect (Hoheisel et al, 1990). Thus or ^'^'C for C substitutions could be made at non­ degenerate positions to increase the overall stability of an oligo for hybridization. Also hybridization in tétraméthylammonium chloride (TMAC) has been shown to eliminate the preferential melting of A-T versus G-C base pairs (Wood et al, 1985), causing the stringency of hybridization to be a function of probe length rather than base content, thus making the melting temperature of degenerate oligos much more uniform amongst multiple heterogeneous