METODOLOGIA PARA EL ANALISIS DE LA INFORMACION

Regions sequenced and primer design

The ligand-binding domain of daf-2 is composed of exons 6 , 7, 8 and 9 while the kinase

domain consists of exon 14. Primers were designed for these regions using Primer 3 software^ and were BLAST^ searched to check that there would be no cross-specificity.

Due to the relatively small size of exons 6 , 7 and 8 , it was possible to design intronic

primers to amplify each exon in its entirety. Exons 9 and 14 are larger, and it was necessary in this case to split each exon into 4 fragments (la-2b) using intronic and exonic primers (Appendix E.2). 0.05 scale oligonucleotides were ordered either from MWG or from Sigma-Genosys Ltd.

Primer dilution

Upon receipt, primers were dissolved in MilliQ HjO by adding a volume in pi equivalent to the mass of primer in pg. They were then further diluted 20:80 primer: MilliQ HjO and were used at this concentration. All primers were stored at -20°C.

DNA extraction

Lysis buffer was prepared in sterile MilliQ water, autoclaved and stored at -20°C: 50mM KCl lOmM Tris (pH 8.3) 2.5mM MgClj 0.45% Nonidet P-40 0.45% Tween 20 0 .0 1 % gelatin

Just prior to use: proteinase K was added at 60pg per ml

8-10 worms were placed onto an OP50-free plate and allowed to crawl away from the bacteria transferred along with them. The worms were then added to lOpl of lysis buffer in the lid of a PCR tube. The lid was placed onto the PCR tube and the droplet spun to the bottom in a microcentrifuge (5 second spin). The tube was left at -70°C for ten minutes in order for lysis to occur, and then placed in a PCR machine using the following cycle:

60°C 1 hour 95°C 15 minutes 4°C hold

Polymerase Chain Reaction

The following were added to a 200^1 PCR tube:

15|il 2x ReddyMix (AbGene) containing 1.5mM MgCl2

11.5|la1 sterile dH2 0

2.5|il DNA

lp,l of each primer

Samples were then cycled on a PCR machine as follows:

94°C 30 seconds (1 cycle)

94°C 30 seconds 1

55°C 30 seconds }" (30 cycles)

72°C 40 seconds J

4°C (hold)

Samples were stored at -20°C until needed.

Running samples on agarose gels

In order to verify that specific amplification had been achieved, a proportion of each sample was run on an agarose gel beside a 1Kb DNA ladder. 1% agarose was prepared in IxTBE. Ip-l ethidium bromide was added per lOOpl gel. The gel was poured into the electrophoresis tank to the indicated level and allow to set, using the appropriate comb to create the required number of wells.

The gel was then loaded with:

lOjLil 1Kb DNA ladder (AbGene) 7pi PCR sample

For larger gels, a DNA ladder was loaded on each side of the samples to aid identification of band size should the gel warp. Gels were run at lOOV for approximately 30 minutes, then the sample bands viewed using a UV trans-illuminator.

Cleaning PCR products

PCR products were made back up to 30p.l using MilliQ HjO, and split into two 15|l i1

aliquots each in a 200pl PCR tube. 15p-l of Microclean (Microzone Ltd) were added to each 15|il sample, mixed well and left at room temperature for 10 minutes.

Samples were microcentrifuged at 13000rpm for 10 minutes in an Eppendorf 5415D microcentrifuge. The PCR tubes were placed in a rack, which was inverted over tissue on a plate-carrying centrifuge rotor. The samples were gently centrifuged at 35G for 1 minute in order to draw off the supernatant, leaving a pellet of cDNA at the base of the tube. Tubes were re-spun if necessary, to remove all the supernatant. 150p,l of 70% ethanol was added to the samples, which were then spun in the microcentrifuge at 13000rpm for 10 minutes. Again, samples were spun upside-down over tissue at 35G for 1 minute in order to remove supernatant. Samples were then dried without lids on a PCR machine at 65°C for five minutes.

Sequencing reaction

5.5|il of MilliQ HjO was added to each dried sample. Enough stock mix was then prepared for all the samples, with the following volumes per sample:

5pi Better Buffer (Microzone Ltd)

Ipl Big Dye Termination Mix (AbGene) (photosensitive)

2pl MilliQ H2O

8 pl of this mixture was added to each sample, along with 1.5pl of the relevant primer

(see above). For each exon fragment, both DNA strands were sequenced by adding the reverse primer to one sample and the forward primer to the other.

Samples were then cycled in a PCR machine using the following program:

96°C 10 seconds 1

55°C 5 seconds \ 25 cycles

60°C 4 minutes J

4"C hold

80pl of 80% isopropanol was then added to each sample, mixed well and left at room temperature for 10 minutes. During this time, samples were transferred to large 1.7ml Eppendorf tubes.

Samples were spun at ISOOOrpm for 10 minutes, and the supernatant carefully poured off onto a piece of tissue. 150p,l of 70% isopropanol were then added to each tube, followed by a further spin at ISOOOrpm for 10 minutes. The supernatant was poured off as before. Samples were then left to dry with lids off at 65°C on a hot-plate for 10 minutes, and stored at -20°C. Samples were prepared for sequencing by A. Smith (UCL Sequencing Technician). lOjLil of high purity formamide was added to each tube, mixed well and heated at 65°C for five minutes. Samples were then transferred to 96-well plates, heated at 96°C for four minutes then cooled on ice for five minutes, after which time they were loaded into an AB13100 capillary gel sequencer.

Sequence Analysis

Sequence data were downloaded and opened in ABI PRISM™ Edit View ABI Automated DNA Sequence Viewer v 1.0.1 (Perkin Elmer). Nucleotide charts were converted into text format, and opened in EditSeq (DNASTAR®). Sequences obtained using reverse primers were reverse complemented so they could be aligned with both wild type and with the sequence obtained using the forward primer. The MegAlign program (DNASTAR®) was then used to align sequences for comparison. Resulting alignments were then checked for nucleotide differences from wild type occurring on both strands. If a potential mutation was detected, the position of that nucleotide was

reliable (i.e. there was a narrow, tall peak on the chart). Sequence regions containing putative mutations were converted into amino acid sequences using the "Translate DNA" option in EditSeq, and the putative amino acid change identified.

B .l l Preparation of microscope slides

Several experiments involved viewing of animals under high power microscopy. To prevent damage, animals were placed on an agar pad on top of a microscope slide, prepared as follows. A tube of fresh, sterile 5% agar (containing no peptone) was melted and kept molten on a 65°C hotplate. One or two drops of the agar were placed using a sterile glass pipette onto a baked microscope slide. The slide was surrounded by two others, along the length of which had been placed a layer of tape. A fourth microscope slide was gently placed on top of the agar drop and allowed to rest on the outer slides. This resulted in the agar being flattened into a pad of a consistent thickness. After approximately 15 seconds the top slide was gently slid off, leaving a set pad of agar.