Heteroduplexes

Delwart EL, Sheppard HW, Walker BD, Goudsmit J, Mullins J1 (1994) Human immunodeficiency virus type 1 evolution in vivo tracked by DNA heteroduplex mobUity assays, J Vhol 68 6672-6683... 

Heteroduplex recombination involves preparing single-stranded DNA from two different homologous genes and mixing them to form heteroduplexes. These heteroduplexes are then transformed into a host that then creates hybrid homoduplexes through in vivo mismatch repair mechanisms [22]. 

Volkov, A.A., Shao, Z. and Arnold, F.H. (1999) Recombination and chimeragenesis by in vitro heteroduplex formation and in vivo repair. Nucleic Acids Research, 27, el8. 

Fig. 4.1. Schematic representation of heteroduplex formation and principle of DGGE. Mutant homoduplexes (M) melt at a lower denaturant concentration than non-mutant homoduplexes (N) and are consequently retarded in the gel. Heteroduplexes (N + M) melt at even lower denaturant concentrations (modified from Borresen, 1996).

Tang, J. and Unnasch, T.R. (1997) Heteroduplex analysis in medical entomology a rapid and sequence-based tool for population and phylogenetic studies. Parasitology Today 13, 271—274. 

Alternatively, the denatured single strands can be made to reanneal to form double-stranded helices. Complementary strands will hybridize to each other. However, if there are sequence differences between two strands, one from each allele, they remain unpaired in the heteroduplex and, as a result, form open loops that reduce migration in the electrophoretic gel. This is the basis of heteroduplex analysis, in which distinct electrophoretic patterns are seen for different alleles, similar to that seen in SSCP (01). 

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. 

This section examines the synthesis of nucleosides that contain seven-membered sugar analogues in place of the deoxyribose component. Nucleosides from the last group have been further incorporated into ONs via solid-phase DNA synthesis. A physical and biochemical investigation of the oligomers thus prepared continues in the next section. The study under review culminated in the assessment of the ability of the oligomers to complex with single-stranded RNA and for the heteroduplexes so formed to serve as substrates of RNAseH. 

DHPLC (C2, L3, U1) uses a proprietary matched ion polynucleotide chromatography column devised by Transgenomics, Inc., in combination with an organic solvent and thermal denaturation (rather than an electrophoretic gel) to resolve heteroduplexes containing a suspected polymorphism from a polymorphism-free homoduplex. Homo- and heteroduplexes are visualized through UV absorbance readings of the eluent as it leaves the column. 

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. 

DHPLC is a useful, high-throughput method to identify novel polymorphisms and to provide genotyping in a variety of situations. It uses PCR to produce a graph of homoduplex and heteroduplex absorbance as these annealed PCR product moieties elute from a column. 

Figure 9.8 Hydrogen bonded duplex consisting of folded strands (a) heteroduplex 10 11 and (b) homoduplex 12 12.

Our results confirmed the feasibility of this strategy (Zeng et al. 2002). Peptide segments were attached to an unsymmetrical, 4-H bonded heteroduplex template with the complementary sequences of ADAA/DADD, leading to four hybrid strands 13a, 13b, 14a, and 14b. Combination of strands 13a and 13b with strands 14a and 14b resulted in four different pairs. The formation of well-defined /3-sheets by different combinations of paired peptide sequences was confirmed by 1-D and 2-D NMR, ITC, and VPO smdies. For example, 2-D NMR (NOESY) showed interstrand NOEs corresponding to protons of duplex templates and the... 

Figure 9.10 Design of supramolecular block copolymers based on the 6-H bonded heteroduplex 15 16. Mixing three templated PS chains with three complementarily templated PEG chains leads to nine block copolymers.

Miranda KC, Huynh T, Tay Y et al. A pattern-based method for the identifieation of microRNA binding sites and their corresponding heteroduplexes. Cell 2006 126 1203-1217. 

DHPLC chromatograms are inspected for abnormal elution of PCR products in comparison to PCR products from genomic DNA with a known genotype. While homoduplexes elute mainly as a single peak at a certain temperature (Fig. 5.2.5), heteroduplexes show two and more elution peaks. 

Fig. 5.2.5 a,b Denaturing high-pressure liquid chromatography chromatograms of two patient samples run at the recommended temperature (60°C) to detect mutations, a Sample without a mutation. The elution peak at 7.8 min represents homoduplexes, b Sample with a heterozygous mutation. In addition to the elution peak at 7.8 min for the homoduplexes an additional peak for heteroduplexes is visible at 7.6 min... 

Jones AC, Austin J, Hansen N, Hoogendoorn B, Oefner PJ, Cheadle JP, O Donovan MC (1999) Optimal temperature selection for mutation detection by denaturing HPLC and comparison to single-stranded conformation polymorphism and heteroduplex analysis. Clin Chem 45 1133-1140... 

Table 8.2.6 Typical conditions for PCR amplification and heteroduplex formation...

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