Especificación de los Casos de Uso

D7S526 and D7S632 are physically linked by YAC yWSS1545 and yWSS922 and their genetic order and orientation was known (figure 3.1 and 3.2). AIu-PCR was used to determine the extent o f overlap between these clones and to detect any links with D7S435 or D7S690 YACs. The ^/w-specific primers utilised in this study were ALEl and ALE3 (Cole et a l 1991; section 2.14.8) which are designed from generally conserved regions fi"om the terminal portions o f the repeat. Amplification is directed out from the ends o f the repeat units. PCR was performed using both ALEl and ALE3 primers in a total volume of 25 pi on YAC DNA solution preparations. This was size fi*actionated through a 3% agarose gel alongside a size marker. Control PCRs using total yeast DNA and no DNA templates were also performed.

PCR with ALEl and ALE3 revealed three bands in common with clones yWSS1545, 922 and 2458 (Figure 3.3). The 1.8 kb product was also shared by yWSS2056, which only contains D7S526. No overlaps were seen with YACs containing D7S435.

The D7S435 containing YACs yWSS29 and 2057 shared three bands (figure 3.3). The fingerprint suggested that yWSS29 is a larger clone as it produced additional unshared products. Alternatively these could have resulted from chimeric ends. Alu-

PCR using single ALE primers did not give clear fingerprints and shared bands were hard to distinguish, therefore PCR was also performed with an end labeled primer and the samples were separated on a 6% acrylamide denaturing gel for better resolution (data not shown). This did not provide any other significant information. Alu-VCK between YACs containing D7S526 and D7S690 failed to show overlaps.

Physical Mapping o f the RP9 Critical Region yj Hind TTT 922 2056 29 31 32 205 2, 332 bp ^ 0 2 7 bp -1,800 bp) 564 bp

Figure 3.3 Alu-PCR fingerprint generated by both the ALEl and ALE3 primers on D7S526, D7S632 and D7S435 containing YACs. The products were separated on a 3% agarose gel. White marks indicate shared bands between YACs. The STSs contained in the YACs are noted above. No overlap was identified between D7S526/632 and D7S435 containing YACs.

Physical Mapping o f the RP9 Critical Region

3.2.2.2 Alu-FC R with D7S690 YACs.

Alu-PCR was performed on a set of 14 redundant YACs (figure 3.2) that

contain D7S690 to establish which YACs extended outwards from the cluster. This information would facilitate generation of a new STS with which to potentially link up with either D7S526 or D7S484/497/460 containing clones (in the proximal region) by chromosome walking (section 3.1.4). Different ^/w-PCR fingerprints were generated by using both ALEl and ALE3 primers together and each primer individually. When analysing the fingerprints generated, it was possible guess the amount of overlap between clones and to determine which extends the furthest. However, bands produced from chimeric or rearranged inserts can be misleading. Only the most prominent bands were scored to avoid confusion from less distinct bands.

The ALEl primer only produced a simple fingerprint of four distinct bands and the level of ghost background was low (figure 3.4). A band of -850 bp was present in all but clones yWSS291 and 2597. yWSS291 did not share any bands with the other YACs. A faint double band of -820 bp and 800 bp was common in clones yWSS3215 and yWSS2597 which indicates that these may extend further out in one direction. The latter clone possessed two additional bands which suggests it may extend even beyond this. This is confirmed by the later assembled STS-based YAC contig across the RP9 interval (Keen et al. 1995a; figure 3.16).

Fingerprints generated from both ALEl plus ALE3 primers were more complex. The number of bands had increased and were not of equal intensity in all YACs, which made scoring of bands more difficult. yWSS291 does share bands with this fingerprint, but no new products are seen in clones yWSS3215 and yWSS2597 (figure 3.5a). It is possible that there is a PCR bias in preferentially amplifying smaller inter-^/w products rather than longer ones, also a large amount of non-specific bands are evident.

Fingerprints generated from the ALE3 primer alone generated at least eighteen products of varying intensity in different YACs. A lack of overlapping bands was seen in YACs yWSS1474, 1787, 4037, 294, 293 and 292 (figure 3.5b). When comparing this with both the single ALEl and the ALEl plus ALE3 fingerprints, the relatively few bands shared among these fingerprints indicated that these YACs are small. Extra less intense bands of -850 bp and 700 bp are seen in yWSS3215 and yWSS2597,

Physical Mapping o f the RP9 Critical Region 1,353 bp 1,078 bp 872 bp 850 bp (-820 & -800 bp) <-600 bp)

Figure 3.4 Alu-PCR fingerprint generated by the ALEl primer only, on D7S690 containing YACs. The -820 bp and -800 bp faint bands generated in YACs yWSS3215 and yWSS2597 suggests that these YACs may extend out further in one direction. yWSS2597 contains two additional bands at -1,100 bp and-950 bp, indicating this may extend even further. This was confirmed by STS mapping (Keen et al. 1995a and figure 3.16). The -600 bp Alu fragment was isolated to generate an STS for chromosome walking.

P h ysica l M appin g o f the R P 9 C ritic a l R egion 4> 4 ' 4 4 / in 1,353 bp 1,078 bp 872 bp 603 bp 310 bp

ill

Figure 3.5 A/w-PCR fingerprints generated by a) both ALEl and ALE3 and b) ALE3 only primers on D7S690 containing YACs. YACs yWSS1474, 1787, 4037, 294, 293 and 292 produced fewer bands in comparison to the others and are probably smaller in size. YACs yWSS3215 and 2597 generated additional products (-850 bp and -750 bp) in the ALE3 only fingerprint. yWSS298 also produced extra bands (-350 bp and -310 bp). STS content mapping confirmed that these clones do extend further from the others.

Physical Mapping o f the RP9 Critical Reg:ion

suggesting that these extend to one side of the cluster. Interestingly, this is supported by STS content mapping analysis which was performed later (Keen et al. 1995a and

see figure 3.16) YAC yWSS298 produced less intense extra double hands of -310 bp and 350 hp. The subsequent STS content mapping data also confirmed that this clone extends distal to the others.