Double-stranded, sequencing templated method

The method of PCR allows selective amplification from a complex genome by enzymatic amplification in vitro. The double-stranded genomic DNA template is denatured by heating, and the temperature is then decreased to allow oligonucleotide primers to hybridize (anneal) to their complementary sequences on opposite strands of the template. The... 

This chapter describes various methods for generating templates suitable for sequencing from PCR products. Three general approaches are discussed (1) the cloning of PCR products, (2) preparation of single-stranded templates from PCR products, and (3) direct use of double-stranded PCR products as sequencing templates. 

Figure 5-21. DNA sequencing by the Sanger-dideoxy method. Single or double stranded DNA is used as a template for a primer-dependent DNA polymerase extension, which is carried out in four separate reaction mixes. In addition to DNA polymerase and the four dNTPs (including an a-radiolabelled nucleoside triphosphate), each reaction mix contains a small proportion of one dideoxy nucleotide derivative, ddATP,...

The isolated clone is sequenced completely. Suitable restriction fragments are subcloned into pUClS plasmid and sequenced using the universal Ml3 forward and reverse primers or specific synthetic oligonucleotides. DNA sequencing is carried out by the dideoxy chain termination method with [ SJdATP, using Sequenase version 2.0 sequencing kit (Amersham) on alkali-denatured double stranded templates. 

Sequence of 2.4.1 strain was already established [18-20] but the amount of mutations with Y strain was unknown. Consequently, the nucleotide sequence of the region which contains the pufL and pufM genes in Y strain was determined by chain termination method of Sanger using base-denatured, double-stranded plasmids as templates. The sequencing strategy employed is summarized in Fig 1 et 2. 

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