Rice Genetics IV - IRRI books - International Rice Research Institute

slip in these regions, producing sequences with varying copies of the repeat.Energy Transfer dye primers (Amersham Pharmacia Biotech, Buckinghamshire,England) have proven useful in these areas, showing higher fidelity and lessslippage, thus allowing longer single-base incorporations. Sequitherm XL(Epicentre Technologies, Madison, Wis.) may also be more processive throughsuch A/T or G/C runs (see Table 1). Use subclones with these chemistries, asPCR will slip. Raising the cycling temperature in the sequencing reactions forGC-rich regions and the use of degenerate oligos are techniques that may beuseful in some cases as well. Thermolabile sequenase used in noncycling sequencingmay also inhibit slippage.Sequencing productsSeparation basedon size2° structureImpropermigrationMissing peaks{Peakcompressions{Fig. 4. Schematic of a typical sequence compression. The altered base spacingseen in sequence chromatograms results from the altered migration of extensionproducts containing secondary bonding structures.Table 1. Widely used sequencing chemistries, including their most useful applications.Chemistry Supplier Advantages DisadvantagesBigDye terminators ABI Even peak heights Frequent sequence stopsNo compressionsdGTP dye terminators ABI Processive through CompressionsG-C rich regions ExpensiveSequitherm XL Epicenter Processive through Expensivehomopolymeric runs4-tube reactionsMust use labeled primerET dye primers Molecular Very processive CompressionsDynamics Even peak heights 4-tube reactionsMust use labeled primer206 de la Bastide et al