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The Chromosome-specific gene library LL09NC01 used in this work was constructed at the Biomedical Sciences Division, Lawrence Livermore National Laboratory,

Livermore, CA 94550 under the auspices o f the National Laboratory Gene Library Project sponsored by the U.S. Department o f Energy.

The LL09 cosmid library was kindly supplied by Dr P. de Jong. LL09NC01 "P ", or LL09 as it is referred to in this thesis, was constructed by flow sorting chromosome 9s from a human/hamster cell line (J640-51) containing, as its only human components, chromosomes 9, 22, and Y. The flow sorted chromosome 9s were partially digested with Mbol, ligated to the Lawrist 16 vector arms and packaged with Gigapack Gold II packaging extracts (Stratagene). The estimated library size was 4 . 2 8 x 1 cfu.

Characterisation of the library by colony hybridisation showed the library to contain 72% Human clones, 23% Hamster clones and 5% non-recombinants. The insert size range is 32-47kb. Further information on the library is available on URL: <http://www- bio.llnl.gov/genome/html/cosmid.html>.

All clone names mentioned in this thesis have a source identifier, given as a prefix, for example "PAG: 12D3". Clones from the LL09 library do not have any prefix. The library was available in two formats; gridded dots on filters for hybridisation and pools of DNA for analysis by PCR.

2.1.7.1 Gridded filters of LL09 library

The library was gridded onto 10 Nylon filters, each comprising 3072 DNA "dots", the "dots" are arranged in "groups" of 8 "dots" each. The arrangement of "groups" and "dots" is shown in Figure 2.1.7 below. When positive clones were scored, their position

was given by taking the filter number (fn)(l-10), then the 384well coordinate of the colony (A1-P24), and the group dot number (gdn)(l-8). Therefore the coordinate of the signal shown as a solid dot on the filter, numbered 1, in Figure 2.1.7 translates to 1P14- 3. In order to derive the original clone number, two more pieces of information were needed, the quad-density number (qdn)(l-4) and the translated 96 well coordinates. The coordinates were easily derived by examining the numbering in Figure 2.1.7, P14 in the 384 well format corresponds to H7 in the 96 well format, this is the actual coordinate of the clone. The quad density number is derived by comparing the group position to the pattern shown in Figure 2.1.7, therefore the quad density number for 384 well

coordinate P14 is 4. This gives us the filter number (fn) 1, the 384 well coordinate P I4, the group dot number (gdn) 3, the quad density number (qdn) 4 and the 96 well

coordinate H7. The clone number is derived by: ((fn-1) x 32) + (gdn x qdn) = ((1-1) x 32) + (3 X 4) = 12 combine this with the coordinates and the cosmid name = 12H7. Since this conversion process would need to be repeated for every signal detected, a program was written for PCs to simplify the process. The Coordinate Conversion Program (CCP), is described in 2.1.8.

Figure 2.1.7 Representation of the filter layout of the LL09 gridded library

The filter dimensions are approximately 80 x 110 mm and the labels shown here highlight the correspondence between 96 well, 384 well and 3072 dot formats. 10 1 2 < C = ' 9 6 W E L L ^ A s ^ B 2 3 4 5 o o o o o o ) 0 0 o o o 0 0 0 B >00 G H I o o o o o o o o o o o o K M N O O O O O O O OOO 0 0 0 o o o o o o o o o 9 6 W E L L 3 8 4 W E L L 1 6 1 7 1 8 1 9 2 0 21 2 2 2 3 o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o I I I I I I I I I I I I I I I o o o 000o o o I I I I I I I I I I I I I I I o o o o o o o o o o o oI Û I I I I I I I I I I I I I I I o o o o o o 000 o o o o o o I I I I I I I I I I I I I I I 000 o o o o o o o o o o o o o o o I I I I I I I I I I I I I I I o o o o o o o o o o o o o o o i l l i l i I I I I I I I I I o o o 0 0 0 o o o o o o o o o I I I I I I I I I I I I I I I o o o 0 0 0 o o o o o o l o o I I I I I I I I I I I I i l i o o o o o o I I I I I I I I I I I I I I I I I I o o o o o o 000 o o oI I I I I I I I I I I I I I I I I I o o o o o o o o o o o o I I I i l l I I I I I I I I I i l i 0 0 0o o o I I I i l l I I I i l i I I I i l i 000 o o o I I I I I I I I I I I I I I I i l i ; ° ° ° ° ° ° I I I I I I I I I I I I I l i I I I 1 o o o o o o 1 o o o o o o I I I I I I I I I I I I I I I I I I 1 o o o o o o 1 2 3 4 Collection o f 4 groups showing quad-density numbering

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A single group comprising 8 dots at coordinate P14 in the 384 well format

2.1.7.2 DNA pools of LL09 library

The library was available for PCR in a series of DNA pools designed to minimise the number of PCR reactions required to search the whole library for positive clones. The pool was divided into 18 superpools, each superpool containing DNA for all the clones in 16 plates, the arrangement of plates in the superpools is shown in Table 2.1.4. Each superpool was divided in turn into 20 subpools called 1-12 and A-H, these were created by pooling all the columns (1-12) and rows (A-H) for all 16 plates in the superpool. A positive result in the subpool required two signals, one in the column pools, 1-12 and one in the row pools, A-H, this indicated the coordinate of the positive clone in the 16 plates making up the superpool. In order to get to the desired clone, the given

Table 2.1.4 Arrangement of LL09 plates in superpools.

Super pool plate numbers in pool

super pool plate numbers in pool 1 1-16 2 17-32 3 33-48 4 49-64 5 65-80 6 81-96 7 97-112 8 113-128 9 129-144 10 145-160 11 161-176 12 177-192 13 193-208 14 209-224 15 225-240 16 241-256 17 257-272 18 273-288