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In-situ hybridisation of fluorescence-labelled cosmids to normal metaphase spreads will allow their position on chromosome 13 to be visualised. The 3' Alu probes were all under 1 kb in length and successful FISH experiments depend on the probes being at least 5 to 10 kb long. Hence, the 3' Alu sequences had to be used to identify longer stretches of genomic DNA that could be used as probes for FISH. Each of the eight 3' Alu probes that were thought to be unique by Southern hybridisation was used to screen a chromosome 13 specific cosmid library. The ICRF library consisted of 20,000 cosmid clones, in the Lorist 4 vector. The 20,000 clones were gridded in arrays of 6x6 on a 20 cm x20 cm hybridisation membrane (Lehrach and et al, 1990). Each filter contained approximately five genome equivalents of chromosome 13. In order to be able to accurately identify the position of a potential positive clone on the filter, the membranes were co-hybridised with Lorist 4 arms which had been radio-labelled with 35s dATP as described in section 2.10. The labelled cosmid vector gave faint signals from each of the colonies on the membrane. Figure 3.16.a shows that, when the 3' Alu probes were labelled with 32p dCTP, they gave dense black dots that were easily distinguished from the background of 35g signal.

For each probe, a series of potential positive clones were identified and then a two stage procedure was used to select those clones which actually contained the correct sequence used in the original hybridisation. Firstly, stab cultures of each of the potential positive clones were obtained and colony purified by streaking out on LB agar plates with kanamycin selection. Individual colonies were grown in arrays on LB agar with kanamycin and transferred to hybridisation membranes as described in section 2.9.4. The membranes were probed with the appropriate 3' Alu clone, those cosmid colonies that yielded strong hybridisation signal were analysed further. Cosmid DNA was prepared from individual colonies using the standard mini-prep procedure (section 2.2.4a). The resulting cosmid DNA was then digested with Eco R1 and separated by electrophoresis on a 1% agarose gel. The gel was blotted and probed with the 3' Alu sequence that had been used to identify the cosmid.

Figure 3.16 shows the typical results obtained by screening the library, in this case with 3' Alu 77. Figure 3.16.a is a photograph of a section of the library filter showing the positive clones showing up against a fainter background of host signal. The Eco R1 digest of the 7 potential positive clones identified is shown in figure 3.16.b. and it can be seen that a multi-band pattern is obtained. Although each of the cosmids shares at least one band of identical size with each of the other cosmids, the overall fingerprints of the seven cosmids are very different. Cosmids 2 and 6 in figure

3.16.b share four bands of identical size and differ by only one band implying that they may contain very similar human inserts. Indeed, as can be seen in figure 3.16.C, of these 7 clones, only cosmids 2 and 6 gave any signal when hybridised with 3' Alu 77.

The FISH data was kindly obtained by two co-workers in the ICH laboratory; Ms. L.A. Hawthorn and Ms. H. Kemp ski and confirmed by Dr. D Shapiro from St. Jude’s Hospital, Memphis, USA. Table 3.2 summarises the results of screening the chromosome 13-specific cosmid hbrary with the eight markers derived by Alu-PCR.

Table 3.2

3 ' Alu Probe N° positives on N° positives Identification L ocation by

library screen after T screen number FISH

77 66 71 78 90 169 62 27 12 same as 77 14 18 17 212 5 same as 62 9 8 3 not analysed 5 C108FO419 C108F0380 C108E055 C108H0710 13ql2 13ql2-ql4 poor signal many centromeres C108A0723 13ql2-q21

Details of the results obtained when the chromosome 13 specific cosmid library was screened with the eight 3’ Alu unique probes. The number of potential positive clones identified by Southern hybridisation is noted as is the number that contained the sequence of interest after two rounds of further analysis as described in section 3.4. The identification number of the clone used as a probe for FISH is given and its chromosomal location by FISH is noted.

& '

Figure 3.16.a. An a u to ra di og ra p h s ho wi n g the results front a t \ p ie a l s c r e e n i n g o f the c h r o m o s o m e 13-speeific c o s m i d librarv. Indi\ idual c o s mi ds are arranged in 6 \ 6 grids which h a \ e been highlighted using labelled Law rist arms. Super i mposed on this we ak signal are the six high intensit) positive clones foll owing h\ bridisation 3' Alu 77 labelled with

d2|\

1 kb 1 2 3 4 5 6 7 1 kb

Figure 3.16.b. An Feo Rl digest o f 7 c o s mi d s identified h\ 3' Alu 77 s h o w i n g multiple bands f r om ea ch c os mi d. Al t h o u g h several c o sm i ds share identical sized bands, the overall 'fingerprints' o f the sev en cosmids

are all different. ^ ^

1kb 1 2 3 4 5 6 7 1kb

f *

rr

Figure 3.16.C. A Southern blot made from the gel shown in figure 3 . 16.b. wa s probed w ith 3' Alu 77. Onlv 2 cosmi ds (arrowed) aetuallv hybridise with 3' Alu 77. the other 5 represent errors in the identification or picking o f the cosmids.

Analysis of the data shown in table 3.2 allowed several important conclusions to be drawn. Firstly, FISH analysis allowed the accuracy of mapping data obtained from the screening of a panel of somatic cell hybrids using hybridisation to be checked. Five of the eight probes (3' Alu 77, 71, 66, 62 and 27) were found to map in 13ql2, as suggested by previous mapping data (figure 3.15). The fact that these five probes all mapped to the region of interest and not to 13p implies that they are likely to closely flank the DG breakpoint. However, 3' Alu 90 appeared to hybridise to the centromeres of many different human chromosomes and not to 13ql2. The presence of this probe in DGF27C11 by hybridisation was not due to its homology to sequences in 13ql2-13qter but to sequences in the centromere of chromosome 1. In this way the data obtained by FISH had allowed more accurate localisation of the probe than was possible by mapping.

Secondly, the pattern of cosmid hybridisation proved to be a useful way of comparing probes. 3 A lu probes that identified identical cosmid clones were very likely to contain the same sequence and, therefore, this data supported the results noted in section 3.3.3b which suggest that 3' Alu 66 and 77 are identical as are 3' Alu 27 and 62.

Thirdly, the number of cosmid clones identified can be a sensitive way of deciding if a given clone is indeed functioning as a unique sequence. The cosmid library has a five times representation of chromosome 13 and, therefore, about five cosmids should be identified with each probe. 3' Alu 169, which identified 212 clones, functions as a low grade repetitive sequence and is not suitable for mapping.

Mapping by FISH was not, however, an unqualified success. 3' Alu 78 did not appear to be an obviously repetitive probe by FISH but the signal obtained was weak and it was not possible to determine the precise location of this clone. Section 4.2.8 discusses the potential reasons for the inability of FISH to accurately map 3' Alu 78 as well as the strengths and weaknesses of the technique.