Single-strand annealing

Figure 22.19. Mechanistic models for the formation of chromosomal aberrations. Regions of sequence homology (e.g., repeats and pseudogenes) are shown as black boxes with the arrow indicating orientation. HR, homologous recombination NHEJ, nonhomologous end joining SSA, single-strand annealing.

Nonhomologous endjoining Homologous recombination Single-strand annealing... 

Fig. 14.3a-d. DSB repair pathways in mammalian cells, a Nonhomologous endjoining (NHEJ), b homologous recombination repair (HRR), c single-strand annealing (SSA), d repair of replication associated DSBs by homologous recombination... 

If DNA from two different species are mixed, denatured, and allowed to cool slowly so that reannealing can occur, artificial hybrid duplexes may form, provided the DNA from one species is similar in nucleotide sequence to the DNA of the other. The degree of hybridization is a measure of the sequence similarity or relatedness between the two species. Depending on the conditions of the experiment, about 25% of the DNA from a human forms hybrids with mouse DNA, implying that some of the nucleotide sequences (genes) in humans are very similar to those in mice. Mixed RNA DNA hybrids can be created in vitro if single-stranded DNA is allowed to anneal with RNA copies of itself, such as those formed when genes are transcribed into mRNA molecules. 

An amplification system that has been successfully commercialized is called the ligase chain reaction (B4). The basis for this reaction lies in using two small single-stranded DNA probes of 10 to 20 bases in length to anneal to target DNA and an enzyme called DNA ligase to link the two probes. Twenty to 50 cycles of this reaction can yield sufficient amplified product. 

Denatured single-stranded DNA can be renatured (annealed) if the denatuiit condition is slowly removed. For example, if a solution containing heat-denatured DNA is slowly cooled, the two complementary strands can become base-paired again (Figure I-l-l 1). 

DNA probes are radioactively labeled single-stranded DNA moleoiles that are able to specifically hybridize (anneal) to particular denatured DNA sequences. Different kinds of probes have been developed for the recognition of particular genes (Table 1-7-2 and Figure 1-7-8). 

The complementary strands are mixed in 1 1 ratio and annealed by heating to 80 C followed by slow cooling (Protocol 15.2). The annealed double-stranded oligomer can be further purified by ion-exchange chromatography (Protocol 15.3) to remove single strands. Protocols 15.1 and 15.3 are modified from the methods described by Aggarwal (1990) and Joachimiak et al. (1987). 

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