The two techniques involved different starting DNA amounts, PCR conditions, agarose gel concentrations, sample placement on the gels, lane size, number of lanes per gel, and duration of electrophoresis.
We used GeneScan analysis software, an internal lane size standard, and an ABI PRISM 377 DNA sequencer.
Genescan 600-Liz (PE Applied Biosystems) was used as an internal lane size standard.
An internal lane size standard was added to the PCR samples for accurate sizing of alleles and to adjust for run-to run variations.
Sixteen primer pairs correctly amplified with the expected size and were run on an ABI 3730 automated sequencer (Applied Biosystems, Foster City, California, USA) using LIZ 500 as the internal lane size standard.
Allele sizing was carried out by automated fluorescent scanning detection in an ABI 377XL DNA sequencer (Applied Biosystems) using ROX500 as internal lane size standard, and the software GENESCAN and GENOTYPER Applied Biosystemss).
For Western blot analysis, undepleted plasma proteins (20 μg) were loaded onto each lane, size fractionated by SDS-PAGE, transferred to PVDF membrane (Amersham Pharmacia Biotech), and blocked with PBS/5% skim milk/0.01% Tween 20 for 30 min at room temperature.
PCR products were diluted 1 10 with water purified by high performance liquid chromatography, and 1.0 μL of diluted DNA was mixed with 13.7 μL of HiDi formamide (Applied Biosystems, Foster City, CA, USA) and 0.3 μL of GeneScan-600 LIZ Size Standard (Applied Biosystems) as internal lane size standard.
PCR products were multiplexed and 1 μL of the diluted mix was added to 8.75 μL Hi-Di formamide and 0.25 μL of an internal lane size standard (Genescan™ 400HD-ROX Standard; Applied Biosystems) and run on an ABI 3130xl Genetic Analyzer (Applied Biosystems), following the manufacturer's protocol.
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Samples were prepared for genotyping by combining 0.7 to 1.0 uL of the diluted amplicons with 8 uL diluted GeneScan ROX500, the internal-lane size standard.
