DNA, heteroduplex

Jaskelioff, M., Van Komen, S., Krebs, J.E., Sung, P., and Peterson, C.L. (2003) Rad54p is a chromatin remodeling enzyme required for heteroduplex DNA joint formation with chromatin. J. Biol. Chem. 278, 9212-9218. 

In CSGE, mildly denaturing solvents in an appropriate buffer can accentuate conformational changes produced by single-base mismatches in heteroduplexed DNA. This increases the differences in electrophoretic mobility between heteroduplex and homoduplex. 

This set of methods makes use of specific enzymes to recognize mismatched base pairs in heteroduplexed DNA strands. In general a test sample is amplified and heat denatured in the presence of a normal control sample. This combination of test and normal control samples is allowed to slowly reanneal forming a mixture of homo and heteroduplexes. The mixture is then exposed to any one of a number of enzymes, which recognize mismatched base pairs at various efficiencies and the sample is then analyzed for these recognized mismatches. There are several types of enzymes and assays available which purport to recognize mismatched base pairs in a heteroduplex or secondary structures formed by mismatches. A short selection of these enzymes and assays are listed below ... 

Diverse analyses demonstrated that we prepared heteroduplex DNA fragments of 7000-9000 base pairs (bp) with complete substitution of both pyrimidine nucleotides [54]. Although the labeled DNA differs very much from natural DNA (for example, with respect to solubility, melting behavior, and circular dichroism), we... 

Each reaction mixture contains the heteroduplex, DNA polymerase, a mixture of the four dNTP species (N = G, A, T, or C), and a small amount of one of the four dideoxynucleotide triphosphates (ddNTP), where no 3 -OH group is present. The ratio of dNTP/ddNTP is high, about 1200 1. DNA polymerase sequentially extends the primer with bases complementary to those present on the opposite strand, but stops when one of the ddNTP species is incorporated. This yields a mixture of fragment lengths, all labeled, where polymerization has stopped at one... 

The second critical step in homologous recombination is the isomerization and resolution of the two DNA molecules. The physical location where all four heteroduplex DNA strands meet, each base-paired with the complementary strand, is called a Holliday junction (named after Robin Holliday, who first proposed it). Figure 23.7 schematically illustrates how these DNA gymnastics are thought to occur, resulting in the formation of DNA characteristic of a homologous recombination event. Starting with the structure at the junction,... 

Figure 25.19 Generation of progeny phage containing two genotypes by replication of heteroduplex DNA. 

Figure 26-6 (A) Electron micrograph of a heteroduplex DNA molecule constructed from complementary strands of phages A,b2 and Ximm434. In AJb2 a segment of X DNA has been deleted producing a deletion loop (labeled b2) and in Ximm434 a piece of DNA from phage 434 has been substituted for X DNA resulting in a "nonhomology bubble" (labeled / f). The...

Replication of a heteroduplex DNA species, i.e., one which contains the desired mutant sequence opposite the wild type strand, will give rise to both mutant and non-mutant progeny, thus lowering mutational efficiency. Several methods have been developed to yield a high frequency of mutations. All have in common that in some manner the wild type plus strand of the heteroduplex DNA is either prevented from being expressed (12-15) or destroyed (16-19). 

The nicking reactions are usually carried out under standard conditions described by the commercial suppliers. The amount of enzyme required to produce nicked DNA ranges from 3 to 17 units/yg of dsDNA with reaction times typically between 1.5 and 3 hr. It is important to ensure that all RF IV DNA has been destroyed, i.e., converted to nicked DNA (RF II), so that all the heteroduplex molecules can be substrates for the gapping reaction. Should any heteroduplex DNA be transfected a lowering of the mutational efficiency will result. 

Soto, D. Sukumar, S. Improved detection of mutations in the p53 Gene in human tumors as single-stranded conformation polymorphs and double-stranded heteroduplex DNA. PCR Methods Applic. 1992, 2, 96. 

Wetekam, W. Identification of template strand in heteroduplex DNA directing cell-free enzyme synthesis. Molec. gen. Genet. 118, 57-60 (1972). 

Spatz and Trautner (1970) and Trautner et al. (1972) have studied transfection with heteroduplex DNA molecules, which were prepared by anneahng complementary strands of wild-type and mutant SPP 1 DNA. The analyses of the progeny reveals that there is an effective mechanism operating in B, subtilis, which converts heterozygous to homozygous molecules prior to their replication. The same technique has been used to demonstrate that both strands of the SPP 1 DNA equally contribute markers to the progeny. 

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