Rice Genetics IV - IRRI books - International Rice Research Institute

ing annotation must be tagged according to the proposed finishing standards ofthe Bermuda genome meetings and the International Rice Genome SequencingProject (Bentley 1996, Guyer 1998, Finishing standards of the IRGSP athttp://demeter.bio.bnl.gov/Guidelines.html).10. Restriction digests must be performed on every clone prior to final submissionand the data from the digest must be compared to the bands predicted by the insilico restriction pattern of both the clone and the cloning vector. The WisconsinPackage (GCG) contains various programs for DNA sequence analysis,including the Mapsort feature, which shows the location of predictedrestriction sites in your DNA sequence as well as the predicted sizes of thebands following digest with a chosen enzyme. Restriction enzymes will havesites in the cloning vector in which the insert is contained and will thereforeadd bands to the resulting fingerprint. The restriction data must agree with thepredicted data within experimental error (~1 kb in a 10-kb fragment, for example)to ensure that the assembly is correct and that the repeats are correctlysorted. Disagreements between the experimental and predicted fingerprints mayindicate a misassembly and must be noted and evaluated.11. Complete the finisher’s clone submission checklist (see Appendix) to ensurethat the clone meets the proper standards (see “Finishing standards of theIRGSP” at http://demeter.bio.bnl.gov/Guidelines.html) and attach any otherinformation that pertains to the finalization of the clone. Submit the clone forfinal review. The final review should be carried out by an experienced finisher,so that the base changes and assembly manipulation can be checked and confirmed.Techniques for problem areasDifficult sequence regions such as repeats and secondary structures often pose problemsin finishing genomic sequence. A variety of techniques have proven helpful in theresolution of regions of sequence that are refractory to standard sequencing techniques.Physical gapsPhysical gaps are regions in which no template is available that spans the gap. Physicalgaps may result from little or no representation of an area in the subclone libraries(cloning bias), or the presence of repeats and secondary structures can often causephysical gaps. The standard solution for gaps between contigs is PCR. If standardPCR does not sufficiently fill the gap (this is often the case in repeat areas), the followingstrategies may be of use:1. High-fidelity enzymes are now available for such amplifications. These enzymeshave a lower rate of base misincorporation than the standard Taq enzymeand work over larger amplification size ranges. Good results have beenobtained with Klentaq polymerase (Sigma, St. Louis, Missouri), the GC-richPCR kit (Boehringer Mannheim, Indianapolis, Indiana), and Platinum Taq withthe PCR Enhancer System (Gibco BRL Life Technologies, Gaithersburg, Maryland).Use of these enzymes with their modified buffers has allowed specific204 de la Bastide et al