Comparative analysis of the HV1 and HV2 regions of human mitochondrial DNA by denaturing high-performance liquid chromatography.

Denaturing high-performance liquid chromatography (DHPLC) was evaluated as a sequencing-independent means of detecting the presence of sequence differences in pair-wise mixtures of nonconcordant amplicons of human mitochondrial DNA (mtDNA). A total of 920 pair-wise combinations of HV1 and HV2 mtDNA amplicons from 95 individuals were assayed by DHPLC for sequence concordance/nonconcordance. For the 72 combinations of amplicons from different individuals who shared identical DNA sequences, DHPLC assays consistently indicated sequence concordance between the samples. This was in 100% agreement with sequencing data. For the 849 combinations of amplicons which differed in sequence, DHPLC detected the presence of sequence nonconcordance in all but 13 assays to yield 98.5% concordance with sequencing. Thus, DHPLC can be used to detect a diversity of sequence differences (transitions, transversions, insertions, and deletions) in the mtDNA D-loop. Accordingly, DHPLC may have utility as a presumptive indicator of mtDNA sequence concordance samples, as a screen for heteroplasmy/situational mixtures, and as a means for the physical fractionation of the individual contributors to an mtDNA mixture prior to sequencing.

Validation of reduced-scale reactions for the Quantifiler Human DNA kit.

Accurate quantification of DNA samples is an important step in obtaining accurate and reproducible short tandem repeat (STR) profiles. Quantitative real-time-PCR has improved the speed and accuracy of DNA quantification over earlier methods, albeit at significantly greater cost per reaction. Here, the performance of reduced volume (10 microL) DNA quantification assays using the Quantifiler Human DNA Quantification Kit was evaluated using commercial standards and single source biological stains (e.g., venous blood, saliva, and semen). In addition, casework-type samples including those subjected to environmental contaminants containing PCR inhibitors and samples having undergone extensive DNA degradation were also quantified. The concentration of DNA in various forensic samples ranged from 0 to 2.9 ng/microL depending on sample source and/or environmental insult. Compared to full-scale reactions, reduced volume assays displayed equivalent to improved amplification efficiency and sample-to-sample reproducibility (+/-0.01-0.17 C(T FAM)). Furthermore, the use of data from reduced-scale Quantifiler reactions facilitated the accurate determination of the amount of sample DNA extract needed to generate quality STR profiles. The use of 10 microL-scale Quantifiler reaction volumes has the practical benefit of increasing the effective number of reactions per kit by 250%; thereby reducing the cost per assay by 60% while consuming less sample. This is particularly advantageous in cases of consumptive testing.

Resolving mtDNA mixtures by denaturing high-performance liquid chromatography and linkage phase determination.

Mitochondrial DNA (mtDNA) sequencing can provide crucial information to forensic investigators when the quantity and quality of DNA would otherwise be limiting. The difficulty of analyzing mtDNA mixtures, however, has been a significant obstacle to its broader use in forensics. Denaturing high-performance liquid chromatography (DHPLC) in combination with direct sequencing makes it possible to determine the linkage phase of individual amplicons in a mixture. The reliability of the approach is based, in part, on the strong correlation between a change in the relative quantities of different DNA amplicons in a given mixture versus a change in the relative electrophoretic peak heights at mixed base positions. Using standard operating procedures previously validated for use in forensic laboratories, this approach enables sequence-specific fractionation of natural (heteroplasmic) or situational (multi-contributor) DNA mixtures prior to direct sequencing. As a novel approach for the rapid and accurate analysis of DNA mixtures, DHPLC may aid criminal investigators by making it possible to obtain definitive mitochondrial DNA results from otherwise challenging samples.

Communication of stabilizing energy between substructures of a protein.

Thermodynamic communication between protein substructures has been investigated by determining the stabilizing effect of mutations at position 52 in the least stable, N-yellow, substructure of cytochrome c on the second least stable, Red, and most stable, Blue, substructures of the protein. A Lys 73 --> His (H73) variant of iso-1-cytochrome c, containing these mutations was used to measure the stability of the Red substructure of cytochrome c through the pH and guanidine hydrochloride (gdnHCl) dependence of the His 73-mediated alkaline conformational transition. The stability of the Blue substructure was measured by global unfolding with gdnHCl and increased by 1 to 3.5 kcal/mol versus the H73 variant. The data demonstrate that the increase in stability of the Red substructure is similar to the increase in global stability, consistent with upward propagation of stabilizing energy from less (N-yellow) to more stable (Red and Blue) protein substructures. The result also supports sequential rather than independent unfolding of the N-yellow and Red substructures of cytochrome c. The data indicate that a leucine at position 52 alters the nature of partial unfolding of the Red substructure, a surprising effect for a single-site mutation. For all variants, the thermodynamics of formation of the Lys 79 alkaline state, which does not unfold the entire Red substructure, shows less stabilization of the portion of the protein unfolded relative to the stabilization of the Blue substructure, indicating that propagation of energy between substructures is somewhat disrupted when unfolding does not correspond to a natural substructure.

Separating human DNA mixtures using denaturing high-performance liquid chromatography.

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.