The crystal of Cas9 shows that it is a bi-lobed structure in which the central nucleic acid recognition (REC) lobe (that consists of bridge helix, Rec1, and Rec2 domains) along with NUC lobe (which consists of RuvC, the HNH, and the PAM-interacting domains) forms a channel to accommodate the negatively charged sgRNA-target DNA heteroduplex
 (Figure 4).
However, none of these approaches are of high resolution nor do they investigate heteroduplex
detection critical for solution genotyping used in high-resolution melting.
Identification of heteroduplex
bands of dihydropteroate synthase gene by CSGE
The amplicons were re-annealed to form a heteroduplex
DNA structure after denaturation.
Tiecke et al (6), identified this mutation by heteroduplex
screening in two unrelated patients with severe clinical involvement, who were not members of their initial screening group.
Recognition of single base replication errors is performed by the MutS[alpha] (MSH2-MSH6 heteroduplex
) or MutS[beta] (MSH2-MSH3 heteroduplex
), excision of the lagging strand from the mismatch by one of the MutL complexes (mainly MutL[alpha] formed by MLH1/PMS2) recruited by MutS protein, and resynthesis of the excised-DNA and ligation by DNA polymerase delta and DNA ligase I .
Detection of hemi/homozygotes through heteroduplex
formation in high-resolution melting analysis.
Although, investigators have applied different methods (Gene Scanning and Heteroduplex
gel electrophoresis) to detect rearrangements, both approaches have similar sensitivities in clonality PCR assays .
In case of ITD-mutant exon-11 of c-kit, the PCR product of mutant exon-11 usually consists of 3 subsidiary bands of 191 bp heterogenous normal alleles, 250 bp mutated alleles, and the heterodimerization (heteroduplex
) between normal and mutant alleles as the largest fragments [13, 14].
TCR beta variable genes (TCRBV) were amplified, after RNA isolation and cDNA synthesis, using TCRBV-specific primers; then PCR products were subjected to heteroduplex
In order to confirm the polymorphisms within genome specific genes, the heteroduplex
hybridization digestion pattern was compared to the ones obtained in each single genotype.