Primed synthesis

The oligonucleotide population is then hybridized to single-stranded DNA and primed synthesis is carried out as described above. 

Prime Synthesis, Inc. offers CPG with pore diameters of 350, 500, 1000, and 3000 A. Their CPG is coded as Native-xxxxx-CPG, where xxxxx is the median pore diameter. 

Figure 36-15. The RNA-primed synthesis of DNA demonstrating the template function of the complementary strand of parental DNA.

DNA synthesis occurs in both directions at each of the rep-licating forks. Once a DNA strand has been primed, synthesis toward the replicating fork can be visualized as continuous. Growth of the opposite,... 

Sanger, F., and A. R. Coulson, A rapid method for determining sequences in DNA by primed synthesis with DNA polymerase. J. Mol. Biol. 94 444-448, 1975. 

The forerunner of the primed synthesis methods was the plus and minus method developed by Sanger and Coulson in 1975 and this marked the first real breakthrough in the search for new and efficient ways of sequencing DNA. This method is the subject of Chapter 2. By itself this procedure does have distinct limitations and additional back-up procedures had to be employed to confirm regions of the sequences deduced. One of these, the depurination method originally introduced by Burton and Petersen, proved a particularly useful adjunct and a description of this method is included as applied to the analysis of pyrimidine clusters in the... 

Chapter 4 describes the application of primed synthesis methods to sequencing single stranded DNAs prepared by cloning restriction fragments into derivatives of the single stranded DNA phage... 

DNA polymerases (like RNA polymerases) are unique among enzymes in that the choice of substrate is determined by the template. E. coli polymerase can copy eukaryotic DNA and animal polymerases can copy bacterial DNA sequences given the appropriate DNA template. This means that DNA sequences, irrespective of their origin, can be accurately copied using E. coli DNA Pol I and this is the basis of the primed-synthesis DNA sequencing methods. 

In these procedures the DNA fragment to be sequenced is initially labelled, at either the 3 - or 5 -ends with [32P] and the DNA chain is cleaved by a set of base-specific reactions to yield families of end-labelled fragments which are separated from each other by electrophoresis on sequencing gels, in exactly the same way as the primed-synthesis fragments are identified. 

This is described briefly since it is now the vehicle of choice for obtaining single-stranded recombinant DNA molecules for sequencing by the primed-synthesis method described in Chapter 4. 

Principle of the plus and minus method (i) Primed synthesis... 

Detailed protocol for the plus and minus primed synthesis method... 

In the ribosubstitution method these problems are circumvented by the addition of one or more ribonucleotides between the DNA primer and the radioactive cDNA. This site is susceptible to cleavage with ribonuclease or alkali. This method can also be used in conjunction with other primed synthesis methods for DNA sequencing (Barnes, 1978 Sanger, Nicklen and Coulson, 1977). 

Depurination analysis of defined fragments generated by primed synthesis... 

Fig. 2.8. Nearest neighbour analysis and quantitative depurination analysis of a defined product from a primed synthesis reaction. When radioactive dATP (or dGTP) is used in the primed synthesis, depurination analysis will yield pyrimidine tracts each of which terminate in a radioactive 3 -phosphate. Thus only those depurination products which lie 5 -adjacent to the labelled nucleotide will be labelled. Each depurination product will be labelled to the same specific activity thus greatly simplifying the quantitation. Digestion of the labelled product with a mixture of micrococcal nuclease and bovine spleen phosphodiesterase yields the nucleoside 3 -monophosphates. Identification of the labelled products (by paper electrophoresis at pH 3.S) gives the nearest neighbours to the labelled substrate.

The plus and minus method represented a major breakthrough in the development of primed synthesis methods for the rapid determination of DNA sequences. 

Taken all in all the plus and minus method is a powerful technique. However, it does have its limitations and the more recent developments using the dideoxynucleoside triphosphates as specific chain inhibitors have much improved both the speed and accuracy of the method and will usually be the method of choice for primed synthesis sequencing. This is described in the next chapter. 

Currently the dideoxy method probably is the method of choice for sequencing DNA by primed synthesis methods and is generally applicable to any DNA that can be obtained in single-stranded form. In the last two years two important advances have been made which have essentially solved the problem of preparing the DNA template in single-stranded form and these are described in the following Section and in Chapter 4. The first method, which... 

In summary there is no doubt that this represents a powerful and convenient sequencing strategy of general applicability. Where the decision is to use a primed-synthesis method and the phage M-13 cloning approach (described in the next chapter) is to be avoided for any reason, the exolll procedure is likely to be the method of choice. A summarized procedure for chain terminator sequencing is given at the end of Chapter 4. 

Primed synthesis methods applied to DNA fragments cloned into phage M13... 

Primed synthesis methods require that either the primer or the template DNA is in single-stranded form. Sometimes it is possible to physically separate the complementary strands from a duplex DNA but this is not always a straightforward procedure. The methods developed by Maxam and Gilbert (1977) (Chapter 5 of this volume), while eminently suitable for separating single... 

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