Annealing complementary single strands

The PCR depends on the ability to alternately denature (melt) double-stranded DNA molecules and renature (anneal) complementary single strands in a controlled fashion. As in the membrane-hybridization assay described earlier, the presence of noncomplementary strands in a mixture has little effect on the base pairing of complementary single DNA strands or complementary regions of strands. The second requirement for PCR is the ability to synthesize oligonucleotides at least 18-20 nucleotides long with a defined sequence. Such synthetic nucleotides can be readily produced with automated instruments based on the standard reaction scheme shown in Figure 9-18. 

Heteroduplex. An annealed duplex structure between two DNA strands that do not show perfect complementarity. Can arise by mutation, recombination, or the annealing of complementary single-stranded DNAs. 

Complementary, single-stranded RNAs are annealed in vitro to generate the final, dsRNA product (Fig. 22.2, B). This dsRNA may be cleaved by Dicer to generate siRNAs (Fig. 22.2, E) that can participate in gene silencing as illustrated in Fig. 22.1. 

Primer— A short oligonucleotide designed to anneal to single-stranded DNA and from which DNA polymerase can add deoxynucleotide triphosphate (dNTPs) in a complementary fashion to the template DNA. A primer pair flanks the target DNA to be amplified in PCR and creates the specificity of the reaction. 

This reaction scheme has been extended to other reactions of the type I topoisomerases such as DNA relaxation, knotting, and the annealing of complementary single-strand circles (Brown and Cozzarelli, 1981 Dean et al., 1982 Dean and Cozzarelli, 1985). For this mechanism to operate for all type I reactions, the enzyme must act similarly on nicked and unbroken DNA, and be able to pass either single- or double-stranded DNA through the break (though in the latter case it is not excluded that the strands could be translocated one at a time). 

Hybridization, which anneals two complementary single-stranded DNA sequences into a double strand, is detected by attaching a fluorophore molecule to either of the sDNA probe or its complimentary sequence cDNA target. In the absence of any catalyst, the process completes itself under the influence of the random Brownian... 

Fig. 3. A, "Slipped" circle formation by denaturation and annealing of a permuted collection of DNA duplexes. Notice that in the permuted DNA molecules the circular order of genes is constant, but the ends are repetitious. However, different DNA duplexes may have different repetitious ends. Upon annealing, the single strand fragments at either end of the same linear duplex may contain complementary sequences, which can undergo further reannealing to produce a circular duplex molecule. In some cases the single-stranded regions at the ends of the linear duplex will not be complementary and so will not result in circular duplex structures. (From Thomas. 1967. J. Cell Physio ., 70 (Suppl. 1) 13-34.)...

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