SSRsand Indels

For SSRs and indels, large polymorphisms between 20 bp to 1 kb can be detected using agarose gel elec-trophoresis. Fragment size differences from two to 20 bp are typically detected on polyacrylamide gels. The fragment separation can be performed by elec-trophoresis on non-denaturing polyacrylamide gels and stained with ethidium bromide or silver. One apparatus to perform this task is the vertical sequencer DASG-400 to DASG-600 (CBS SCIENTIFIC, California, USA). The throughput of this system is approximately 600 to 900 data points per day requiring substantial manual labor, although supply costs are low. Higher throughput can be achieved by size separation using denaturing polyacrylamide and a fluorescent dye-based automated detection system. Flu-

orochromes are covalently attached to one of the primersusedinthe amplificationandthe productsare detected via laser excitation of the label. The LI-COR IR2®slab gel-based system (Li-COR, Nebraska, USA) uses two fluorochromes thereby increasing marker throughput to approximately 2,000 data points per day. This system requires manual labor in preparing the gel and loading of samples whereas automated analysis of band fragment sizes is performed using the genotyping software. Other fluorescent-based fragment separation systems utilize capillary electrophoresis technology identical to that used for dideoxy DNA sequencing. Manual labor is minimal when preparing the capillary, loading the samples, and detection, sizing and scoring the fragments. The Beckman CEQ8800® can detect fluorescently labeled fragments for eight or more markers simultaneously ( with a throughput of approximately 1,536 data points per day. Although the throughput is not very high on the Beckman CEQ8800, the system is versatile and able to handle diverse sets of samples from users. The ABI system is another frequently used genotyping platform (Applied Biosystems, Foster City, CA, USA). From 16 (ABI 3100) to 96 (ABI3730xl) capillaries are run simultaneously, which greatly improves throughput. The four fluorochromes used in ABI chemistry and detection increase the number of multiplexed markers to at least 16 per capillary, resulting in daily throughput of 3,072 to 18,432 data points.

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