Rice Genetics IV - IRRI books - International Rice Research Institute

amplification of regions that previously gave either no product or multiplebands. (Note: Because many enzymes have some polymerase activity at 4 °C,false annealing and extension may occur while the reaction mixture is preparedon ice. To reduce the occurrence of this problem, use a “hot start” inwhich the enzyme is the last component added to the reaction mixture in thethermal cycler at 95 °C.)2. Add DMSO (5–8% maximum) to the PCR reaction. DMSO tends to relax theDNA template, thereby facilitating smooth extension. Higher annealing temperatures(55–65 °C) may also be useful.3. Pick longer custom primers (30mers) and move the priming sites around, tryingmultiple combinations from multiple distances.4. Try PCR and/or direct sequencing off of different template types, includingBAC DNA, plasmid subclones, and M13 subclones.Techniques in addition to PCR include using in vitro transposons (see “Techniquesfor problem areas”) and subcloning and subsequently sequencing restrictionfragments that are known to span the gap.Sequence gapsSequence gaps differ from physical gaps in that there are templates available thatreadily cover the gap, but sequencing reactions are hindered and the enzyme does notprocess through the region. To sequence through such regions, it is often necessary toemploy methods other than primer walking, which is both inefficient and sometimesineffective. These gaps may be the result of compressions in the sequence, internalstructures that deter sequence extension, or mono-polynucleotide runs. Possible solutionsfollow:1. Compressions—sequence both strands (compressions in a specific area usuallyreside on only one strand) or use terminator chemistry (the large dye moleculeon the 3’ extension end of the sequence tends to “flatten” compressions).BigDye terminator kits (Perkin Elmer, Foster City, Calif.) have proven quiteeffective at compression resolution. Figure 4 shows a schematic of a typicalsequence compression.2. Hard sequence stops, or regions where the processing of the enzyme is significantlyand abruptly hampered, often occur in GC-rich sequences. The dGTPdye terminator kit from Perkin Elmer (Foster City, Calif.) has proven extremelyuseful in such regions. Most available terminator kits use the inosine analog tohelp combat GC compressions, but it is thought that the lower efficiency ofinosine incorporation coupled with the reduced processing of the enzyme inregions of secondary structure or “hairpins” leads to repressed sequencing, orstops. By replacing inosine with guanine, the dGTP kit enhances processingand allows sequencing to advance through the structure. Compressions may beevident in such regions, but are generally less problematic to resolve. Table 1shows the commonly used sequencing reagents and their uses.3. Mono-polynucleotide strings—mono- and dinucleotide repeats also challengethe effectiveness of typical sequencing chemistries. Dye terminators tend toStrategies and techniques for finishing genomic sequence 205