Separating human DNA mixtures using denaturing high-performance liquid chromatography

Abstract

DNA mixtures represent challenging samples that are rarely amenable to direct DNA sequence analysis and many of the strategies available to separate mixtures are both labor and time intensive. Denaturing high-performance liquid chromatography is an accurate and rapid approach for the detection and scoring of mutations. It can also be used to separate DNA mixtures. The technique relies on the chromatographic separation of crosshybridization products to isolate the individual components of a mixture. By eliminating secondary amplification and excessive manipulation prior to sequencing, denaturing high-performance liquid chromatography can streamline the analysis of conditions ranging from somatic mosaicism, microchimerism and mitochondrial heteroplasmy to evidentiary material containing mixtures of DNA encountered in forensic investigations.

Disease-associated CIAS1 mutations induce monocyte death, revealing low-level mosaicism in mutation-negative cryopyrin-associated periodic syndrome patients

“…[15][16][17] Diagnosis of mosaicism by conventional genomic sequencing using the dideoxy termination method is often difficult, because the overlapping chromatogram of the mutant is easily missed when the frequency of a mutant allele is less than 20% to 30%. 18 Heteroduplex-based methods 15,19 or subcloning-based analysis of mutant alleles enable one to detect such low-level mosaicism; however, these methods are resourceintensive, and cannot distinguish whether the detected mutation is disease-causing or simply a nonfunctional single nucleotide polymorphism (SNP). An alternative approach involves the isolation of mutant cells using functional analyses based on their characteristic biologic features, and then determining the DNA sequence of the isolated cells.…”

Forensic Science

“…Denaturing HPLC has also been proposed as a possible approach to separating mtDNA amplicon mixtures (181,182) as has a mismatch primer-induced restriction site analysis method (183).…”

Comparative Analysis of the HV1 and HV2 Regions of Human Mitochondrial DNA by Denaturing High‐Performance Liquid Chromatography

“…Within an mtDNA sequencing laboratory, screening by DHPLC makes it possible to detect samples that contain mixtures of nonconcordant amplicons immediately after PCR amplification and without having to sequence them. For both heteroplasmic and situational mixtures, this approach would enable the analyst to identify potentially challenging samples and mark them for “special handling”—whether that be use of alternate sequencing primers to avoid C‐stretch polymorphisms or the application of emerging technologies for resolving mixed samples (29–31). …”