Abstract
Publisher Summary This chapter focuses on the sequencing products of polymerase chain reaction (PCR). The method described allows the isolation, purification, and sequencing of double-stranded DNA (dsDNA) templates generated by the PCR. It is applicable to the sequencing of other dsDNA templates, such as restriction endonuclease fragments or plasmid DNA. In performing DNA sequencing, it was found that denaturation by alkali is an effective method for the simultaneous processing of multiple templates. Denaturation by alkali has proved useful for sequencing templates containing repetitive sequences. Methods involving the heat denaturation or production of single-stranded DNA (ssDNA) proved problematic in sequencing such templates. Following ethanol precipitation, centrifugation, and desiccation, individual templates may be stored indefinitely prior to the initiation of DNA sequencing reactions. Because additional nucleic acid species (i.e., additional templates, oligonucleotides, and deoxynucleotide triphosphates (dNTPs) have been removed, templates prepared in this manner are suitable not only for DNA sequencing but also for Southern blot hybridizations, in situ hybridization, and microinjection.
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