Dideoxynucleotide sequencing

Fig. 6.22. The Sanger DNA-sequencing procedure, (a) The Sanger dideoxynucleotide sequencing reaction with 32p. abeled primers, (b) Acrylamide gel separation of the labeled fragments. (Adapted from Watson JD, Gilman M, Witkowski J, et al. Recombinant DNA, 2nd Ed. New York Scientific American Books, W.H. Freeman and Company, 1992 with permission.)...

Figure 26-16 Sanger dideoxynucleotide sequencing of a sample oligonucleotide, called the template strand. The attached known primer sequence tells the enzyme DNA polymerase where to start replication. Replication stops when a 2, 3 -dideoxyribonucleotide n the experiment shown, it is ddA) is added to the growing chain. Eiectrophoresis of the resulting mixture reveals the length of the fragments with the corresponding end nucleotide and, therefore, the position of this nucleotide in the replicate strand and its complement in the template strand.

Yet nucleic acids do it relatively quickly and with amazing precision. Indeed this reannealing (or hybridization) capability between a polynucleotide and a complementary piece of oligonucleotide (commonly called a printer or a probe) forms the basis of such diverse and critical techniques as dideoxynucleotide sequencing, site-specific mutagenesis, cDNA cloning, and hybridization screening of recombinant DNA clones. Nucleic acid hybridization also serves as the basis... 

Reverse transcriptases are also valuable reagents in the direct dideoxynucleotide sequencing of RNA (110). In fact, RTases have been instrumental in obtaining nucleotide sequence information directly from viral RNA genomes, 16S rRNA (111), and mRNAs (112). The higher thermostability of AMV RTase compared with that of MoLV RTase makes the AMV RTase more useful for nucleotide sequencing at elevated temperatures. 

RNA sequencing can he directly performed from dsDNA templates cloned downstream of a phage promoter, T7, T3, or SP6 (30,82). Transcription reactions are performed in the presence of a chain terminator, 3 -deoxynucIeoside 5 -triphosphate, in a way similar to the dideoxynucleotide sequencing of DNA. The difference is that the DNA sequencing by RNA transcription obviates the need to prepare ssDNA templates or to use sequencing primers. 

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