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Electrophoresis through a polyacrylamide gel is an effective means of separating small DNA fragments with high resolution, allowing fragments differing in size by as little as

1 bp to be separated. Denaturing polyacrylamide gels are polymerised in the presence of an agent such as urea which suppresses base pairing in nucleic acids. Denatured (single stranded) DNA migrates through these gels at a rate that is determined by fragment size and almost completely independent of base sequence and composition, permitting sizing of fragments according to distance traveled through the gel. Smaller fragments migrate further than larger ones because larger fragments are retarded more than smaller fragments by the pore size of the gel polymer.

2.5.4.1 Polyacrylam ide gel preparation

36cm well-to-read glass plates were used with a Perkin Elmer 377 automated

sequencer. Plates were cleaned with detergent and rinsed with distilled water. The dry plates were assembled in the 377 cassette prior to pouring the gel. The catalysts TEMED (Sigma) (35 pi) and freshly prepared 10% ammonium persulphate solution (APS) (Sigma) (250 pi) were added to 50 ml of 4% acrylamide gel mix to start polymerisation. The mix was then taken up into a 50 ml syringe and carefully

introduced into the notch between the front and back plates, spreading evenly between the glass plates. A spacer was inserted into the upper notch between the plates and the gel left for two hours to polymerise. After polymerisation, the upper spacer was

removed and a 48 or 64 well shark’s tooth comb was carefully inserted in its place. The cassette and plates were then placed in the 377 sequencer and the plates checked for background fluorescence using Genescan (version 2.0.2) software (Applied

Biosystems). Heating plate and buffer chambers were assembled and 1.3 L of 1 x TEE buffer added before pre-running the sequencer until the gel temperature reached 50^C. Samples were then loaded.

2.5.4.2 Pooling o f PCR products fo r loading

Up to 12 non-overlapping microsatellite markers, amplified from a single DNA sample, were run simultaneously in each lane (multipooled). PCR products from each DNA

sample were first pooled according to the dye they contained as follows: 6-FAM - 4pl;

TET - 4pl; NED - 4pl; HEX - lOpl. These volumes were adjusted according to the yield of the PCR reaction as determined from agarose gel electrophoresis. Pooling was

performed in microtitre plates using an eight channel pipette. 2.5|li1 of pooled product from each well was then aliquoted into a fresh microtitre plate and an equal volume of loading mix added. The loading mix consisted of 100 pi of deionised formamide, 20pl of loading buffer (blue dextran, 50mg/ml, EDTA 25mM, Perkin Elmer) and 24pl of Genescan 350-TAMRA/ 500-TAMRA 400 ROX (if NED-labeled primer used) size standard (depending on the anticipated size of the largest PCR fragment) (Applied Biosystems). The final mix of pooled product and loading mix was denatured at 95^C for 2 minutes in a Hybaid thermal cycler and then placed immediately onto ice before loading.

2.5.43 Gel loading and electrophoresis conditions

Wells were carefully flushed with 1 x TBE buffer immediately prior to loading. Alternate (odd-numbered) wells were loaded with 1.8p.l of final mix using Sorenson MiniFlex 0.2mm flat tips (Anachem). Great care was taken to avoid spill-over into adjacent wells. Electrophoresis at 3,000 V for two minutes ensured that samples were run into the gel before even-numbered lanes were loaded. Loading of alternate lanes made it possible to distinguish adjacent lanes in the final gel image and improved the ability of the software to track lanes correctly. Total run time was two hours. A maximum of 36 samples could be run in adjacent lanes. Where more than 36 DNA samples from a single family were genotyped for a given marker (necessitating two electrophoresis runs), five samples from the first run were included in the second run as a control, to ensure gel-to-gel consistency of scoring.

PCR products produced using fluorescent-tagged primers were sized by electrophoresis through a denaturing 4 % polyacrylamide gel in an automated DNA sequencer (Applied Biosystems, model 377). During electrophoresis, a section of the gel furthest from the loading comb is scanned by a laser causing each dye moiety (attached to one

oligonucleotide primer incorporated into PCR fragments) to emit light of a known wavelength as it migrates past the laser. A size standard consisting of DNA fragments of known size, labeled with the fluorescent dyes TAMRA or HEX, is run in each lane to allow accurate sizing of PCR fragments. This method of DNA sizing has the great advantage over radioactive methods in that markers of non-overlapping size and dye composition may be multiplexed in each lane, maximising efficiency and increasing sample throughput. As many as 24 microsatellites may be run in each lane although in

Figure 2.2: An exam ple o f a fluorescent m arker gel im age analysed using GeneScan. PCR products am plified by fluorescent-labelled prim ers are detected by a laser detection device as they run through a polyacrylam ide gel inserted in an ABI377 autom ated sequencer and are analysed by the associated com puter softw are, G eneScan.

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2.6 DATA ANALYSIS FOR FLUORESCENT-LABELED PCR PRODUCTS