Duplex renaturation

DNA Renaturation Involves Duplex Formation from Single Strands Chromosome Structure... 

Steps in denaturation and renaturation of a DNA duplex. In step l the temperature is raised to the point where the two strands of the duplex separate. If denatured DNA is slowly cooled, the events depicted as steps 2 and 3 follow. In step 2 a second-order reaction occurs in which two complementary strands of DNA must collide and form interstrand hydrogen bonds over a limited region. Step 3 is a first-order reaction in which additional hydrogen bonds form between the complementary strands that are partially hydrogen-bonded (zippering). Once complementary strands are partially bonded, the zippering reaction occurs rapidly. In the overall process, step 2 is rate-limiting. 

Kinetic analysis indicates that renaturation is a two-step process. In the slow step effective contact is made between two complementary regions of DNA originated from separate strands. This rate-limiting step called nucleation is a function of the concentration of complementary strands. Nucleation is followed by a relatively rapid zippering up of adjoining base residues into a duplex structure. The steps involved in denaturation and renaturation are depicted in figure 25.14. 

Renaturation. The process of returning a denatured structure to its original native structure, as when two single strands of DNA are reunited to form a regular duplex, or the process by which an unfolded polypeptide chain is returned to its normal folded three-dimensional structure. 

Hydroxylapatite Hybridization. Hydroxylapatite (HA) is a form of calcium phosphate that preferentially binds double-stranded DNA when in an appropriate concentration of phosphate buffer 21,22 This specificity provides a means for separating renatured DNA duplexes from free-solution reassociation reactions. Brenner et al.21 developed a batch procedure that allows handling of up to 10 samples at the same time, thus greatly increasing the utility of HA for DNA hybridizations. The procedure given here was derived from the work of Brenner et al.21 and Price et al.6... 

It is sometimes necessary to remove rapidly renaturing sequences from DNA samples before measuring the extent of relatedness, and this can be done on HA columns as described by Price et al.6 Self-annealing of the radiolabeled probe DNA can be minimized if the ratio of target to probe is kept to at least 1000 1. It may also be useful to determine the amount of self-annealing to be certain that this does not bias the calculated relatedness. Finally, a simplified version of the HA procedure was described by Lachance24 in which HA-containing minicolumns maintained under controlled temperatures are used to separate the duplexed DNAs. 

The double helix is a relatively stiff and elongated molecule. Consequently, a solution of DNA has a high viscosity. If such a solution is heated to 95°C, the viscosity drops markedly, reflecting a collapse of the double-helical structure. This is known as denaturation and is accompanied by separation of the duplex into its single strands, which are fairly flexible. Denaturation and renaturation provide valuable information on important properties of the DNA obtained from various sources. Denaturation also provides the basis for very precise and sensitive approaches to the identification of specific sequences in both DNA and RNA. This has been central to the rapid developments in molecular genetics. 

Why does circular duplex DNA renature more rapidly than linear duplex DNA ... 

When aqueous solutions of DNA are raised to high temperatures, the two strands of the duplex molecule separate (Marmur and Doty, 1959). Slow cooling of the heat-denatured DNA results in renaturation of the two strands so that many of the properties of the original duplex molecule are restored (Marmur and Lane, 1960 Doty et... 

Fig. 2. The kinetics of reassociation of calf thymus DNA measured with hydroxyapatite. The DNA was sheared to small fragments and the single-stranded fragments incubated under renaturing conditions. At various times samples were passed over a hydroxyapatite column at 60 C, under conditions in which only duplex DNA binds to hydroxyapatite. Because of the large range in Cot values, different initial concentrations (Cq) of calf DNA were used to obtain the complete renaturation curve A, 2 jug/nni , 10/xg/ml o, 600Mg/ml and 8,600 /ug/ml. The crosses are radioactively labeled E. coli DNA at 43 jug/ml present in the renaturing mixture of calf thymus DNA at 8,600 The rate of renaturation of E. coli DNA provides an internal standard with which the...

Fig. 8. A diagram to show how repetitive nucleotide sequences, that are exchanged between the two strands of a DNA duplex, can cause renaturation of the separated single strands. A, the sequence is exchanged between strands and also reversed from 5 to 3 direction in one strand to 3 to 5 direction in the complementary strand. Incubation of the separated strands under renaturing conditions will result In the formation of partially duplex circular structures by the renaturation of complementary regions In the same strand.

Fig. 8B, the repeated sequence is exchanged between strands, but the 5 to 3 direction of the sequence is the same in each strand. Renaturation of the separated strands will result In pairing of the complementary sequences to produce a hairpin structure. Both forms of duplex involve the folding back of the single strands upon themselves, hence the term fold-back DNA. The duplex formed In this way will have similar physical properties to duplex structures formed by the hybridization of two complementary strands, except that the rate of formation of fold-back duplexes will not show a dependence on the concentration of single strands. The sequences that participate in the formation of fold-back duplexes are held together In close proximity by their presence on the same single strand of DNA. 

The stability of a DNA duplex depends on, besides the chemical and physical structure of the DNA itself, many environmental factors such as solvent, temperature, and salt composition and concentration. For example, when a stable DNA duplex is heated to higher temperatures, the double helix structure will be gradually denatured into two coiled single strands. When cooled to room temperature, the two single strands can reassociate to again form the duplex, a process called DNA renaturation (sometimes also called DNA hybridization or annealing). These processes are reversible, as illustrated in the following equilibrium ... 

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