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Generating the cDNAs and Genes of G S T M Ib and GSTN/14

P 1 5-ATGCCCATGATACTGGGGTACTGG-3" (sense) P 2 5-GTGGAGGTCAAGGACATCATAG-3' (antisense) P 3 5-CTATGATGTCCTTGACCTCCACCG-3' (sense) P 3 (M 1 )3 5'-GGAATGAGATCTGTTTTGCTTCAC-3' (sense) P 3 ( M 4 ) b 5'-CACTAGAGTTTTGCCATACATCCT-3' (sense) P 4 5'-CTACTTGTTGCCCGATACATCCAT-3' (antisense)

Determining the G S T M1 Null Genotype

P 5 5'-CGCCATCTTGTGCTACATTGCCCG-3' (sense) P6 5'-A TC TTC TC C TC TTC TG TC TC -3' (antisense) P 7 ( M 1 ) 5'-TTCTGGATTGTAGCAGATCATGC-3’ (antisense) P3 and P4 (see above)

Mapping the GSTM1 and GSTM4 Genes

P 8 ( M 1 ) 5'-GGTACCATTCATCCTCCAAGTGC-3‘ (sense) P 9 ( M 1 ) 5'-GCTTCAATTGGATACATCAGCATT-3' (antisense) P 3(M 4) and P4 (see above)

^ M l: This primer is specific for G S T M1 gene. ^ M4: This primer is specific for G S T M 4 gene.

2 .2 .2 H DNA ligation

Ligation reactions w ere carried out in a total volum e of 2 0 ^1 which comprised as follows: 2 pi of lO x ligase buffer (Appendix 6.3.1 H), 1 - 2 pi of vector DNA and 5 -1 0 pi of insert DNA in LM P agarose gel (the gel would have been melted at 6 8 ° C for 10 minutes, then kept at 37 ° C prior to the ligation), 1 pi of T4 DNA ligase ( 6 units/pl) and distilled w ater to the final volume. The reaction was thoroughly mixed by pipetting to disperse the gel, and incubated at room temperature for at least 2 hours. The reaction was stopped by heating the mixture at 6 8 ° C for 10 minutes immediately before transformation.

2 .2 .2 1 Transformation of E. coli by plasmid DNA

In this project, the efficient introduction of DNA into E. coli has obtained 1 0 ^ -1 0 ^ colonies per microgram of plasmid DNA by either chemical treatm ent or electroporation.

The following rapid and convenient chemical method of treating E. coli has been used to prepare com petent bacterial cells for DNA transformation (Chung and Miller, 1988) (Chung e t al., 1989). A single colony of host strain of E. coli (D H S a or N M 522) was grown in 1 0 0 ml of LB medium (Appendix 2.3.11) at 3 7 ° C with 2 25 rpm shaking until the O D eo o w as 0 .5 (1 O D goo = 8 x 1 0® cells/m l). The cell culture was centrifuged at 3 ,0 0 0 rpm at 4 ®C for 1 0 minutes, the pellet resuspended in 1 0 ml of ice-cold TS B medium (Appendix 2.3.11) and then incubated on ice for 1 0 m inutes. Aliquots (0 .5 ml) of the com petent cells w ere placed in 1.5-m l pre-chilled microcentrifuge tubes on a solid C0 2/ethanol bath and store at -80 ° C . Portions of the competent cells (0.1 ml) with 1 0 0 pg of recom binant plasm id D N A (the volum e of the ligation

1.5-ml pre-cold microcentrifuge tubes and placed on ice for 5 -3 0 minutes. 0.9 ml aliquot of TSB solution with 2 0 mM of glucose was added to each tube and incubated at 3 7 ° C with shaking (225 rpm) for 1 hour.

For transform ation by electroporation (D ow er et al., 1988), 4 0 0 ml of LB medium with 1 / 1 0 0 volum e of fresh overnight grown E. coli cells w ere incubated at 3 7 ° C with shaking (225 rpm) to an ODeoo of 0.5. The cell culture was cooled on ice for 15-30 minutes then centrifuged at 6,000 rpm, at 4 ° C for 15 minutes. The cells were resuspended in succession in 400 ml, 2 0 0 ml of cold distilled water and 1 0 ml of 1 0% glycerol before being recentrifuged. The cells w ere then resuspended in a final volume of 2 ml of 1 0% glycerol (the cells concentration should be about 3 x 10^0) and these electrocompetent cells can be frozen in aliquots on dry ice and stored at -70 ° C . For electroporation the ElectroPorator (Invitrogen) was set at 25 pF and 2 .5 kV, and the pulse controller adjusted to 2 0 0 O . A 4 0 pi aliquot of electrocom petent cells w ere m ixed with 1-2 pi of recom binant plasm id DNA to a cold 0 .2 cm gap electroporation cuvette. The cells were pulsed once at the above settings and added immediately to 1 ml of S O C medium (Appendix 2.3.11). The transformants were incubated at 37 ° C for 1 hour.

Portion (5 0 -1 0 0 pi) of tran sfo rm an ts w ere plated on antibiotics- containing LB-agar(Appendix 2 .3 .1 1) and incubated at 37 ° C for 12-24 hours to allow selection of transformants. If the blue/white screening was available, the plates were spread with 1 0 0 pi of 2% X -g al and 40 pi of 100 mM IP TG (Appendix 2.3.11); these components were allowed to absorb into the agar for 30 minutes at 37 ° C prior to plating cells.

2.2.2J Double-strand DNA sequencing

The sequencing of double-strand DNA is based on the modified chain- termination method (Sanger e t al., 1977) (Hsiao, 1991). The method employed in this thesis used 5pl of mini-prep DNA (about 0.1-1 .0 pg) per sequencing reaction. The D NA w as den atu red with 1 pi of 1 M N a O H in a 0.5-m l microcentrifuge tube and incubated at 3 7 ° C for 1 0 minutes. 1 pi of 1 M MCI, 2 pi of 5x reaction buffer and 1 pi of sequencing oligo-prim er (0 .6 pg/pl) were added subsequently, and mixed well by pipetting. The reaction was incubated at 37 ° C for 1 0 minutes. Follow the remaining steps as described in the Sequenase Version 2 . 0 kit protocol (United States Biochem ical). This sam ple labelled with 2^S can be stored at - 2 0 ° C for 1 week.

A 6% [w/v] polyacrylam ide gel (0.4 mm in thickness) containing 8 M urea and 1x TBE buffer was prepared 2 - 2 0 hours prior to use. The samples were prepared for electrophoresis by heating them at 85 ° C for 2 minutes before loading 4-5 pi onto the gel. The gel was run in 1x TB E buffer at 55 W constant power for 3-4 hours depending on the position of interesting sequences. The gel w as fixed in 10% [v/v] acetic acid and 10% [v/v] m ethanol solution for 15 minutes, then transferred onto a sheet of W hatm an 3M M paper before the gel was dried at 80 ° C with vacuum for 45 minutes. The dried gel was exposed with direct contact onto the Kodak X-O m at AR film at room temperature overnight.

Together with the use of Mn^+ buffer or 4% [w/v] of gel, DNA sequences in the 20 to 400 nt range from the primer can be obtained in one sequencing reaction.