Rice Genetics IV - IRRI books - International Rice Research Institute

4. Structure problems—subcloning is a viable option for areas in which a PCRproduct is readily reproducible, but sequence quality is poor or stunted (cloningindividual PCR molecules and using a universal primer usually produce abetter sequence). (Note: Because of base misincorporation, high-fidelity enzymesmust be used and at least three different concurring subclones on bothstrands are necessary for coverage.) Invitrogen’s TA subcloning kit (Carlsbad,Calif.) has been particularly helpful in obtaining a clean sequence from PCRproducts that were refractory to direct sequencing with nested primers.Small insert libraries (SILs, McMurray et al 1998) are an alternate technique fordisrupting the secondary structures of repeats. In this method, clones spanning theproblem region are sheared into 200–500-bp segments that are subsequently subclonedand sequenced. These small pieces of DNA are generally not subject to the secondaryfolding, which deters sequencing in larger inserts (see Fig. 5).In vitro transposons may also be useful. The Template Generation System(Finnzymes, Watertown, Mass.) uses the Mu transposon to randomly insert noveluniversal priming sites into segments of DNA. Inserting multiple priming sites withinthe problem area tends to break up the secondary structure and allow sequencingto occur from within the problem region. Transposition has been a successfulAlignment containing a gapTemplate containingsecondary structure thatspans the gap regionTemplate sheared intosmall fragmentsFragments subcloned tocreate a small insert library(SIL)Resolution of the gapregion by sequencingsubclones from SIL andassembling sequenceFig. 5. Schematic of the small insert library (SIL) sequencing technique(McMurray et al 1998). Shearing a template covering a difficult sequenceregion into 200–500-bp segments may disrupt secondary structures that oftendeter sequencing.Strategies and techniques for finishing genomic sequence 207