PubMed · 16248478
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Tracy L Paxon, Andrew G Ewing. 2006. Separations in multiple-channel microchips.. https://pubmed.ncbi.nlm.nih.gov/16248478/
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Next-generation sequencing (NGS) provides increased discriminatory power in forensic DNA analysis due to the detection of isoalleles. Differences in sequences between alleles allow for a second layer of differentiation between DNA contributors beyond the number of short tandem repeat (STR) repeat units. However, because NGS is a more time and resource-intensive analysis than conventional capillary electrophoresis (CE), laboratories may benefit from indicators that suggest NGS is likely to provide added value. This study examined whether CE migration offsets, measured as residuals in the OSIRIS analysis software, can differ significantly among STR isoalleles. Residuals represent the time offset between a sample allele peak and its corresponding allelic ladder peak. Paired CE and NGS data from 95 single source samples were analyzed for CE-based residual differences, as the NGS data provided the sequence information of the corresponding isoalleles. Residual values differed significantly among isoalleles at several STR loci. Statistically significant differences were identified at D16S539 and D3S1358, as well as at specific allele lengths within D12S391, D13S317, and D8S1179. These findings demonstrate that CE residual variation can reflect underlying STR sequence differences between contributors. In practice, residual-based metrics could help laboratories to identify casework reference samples where NGS is likely to provide additional discrimination, without the need for processing outside of a routine CE workflow. Due to the potentially large number of isoalleles, community wide efforts to aggregate CE residual differences versus isoallele sequences may be useful in the validation and implementation of this approach to add value to forensic DNA analyses.
CE- and microchip-based separations coupled with LIF are powerful tools for the separation, detection and determination of biomolecules. CE with certain configurations has the potential to detect a small number of molecules or even a single molecule, thanks to the high spatial coherence of the laser source which permits the excitation of very small sample volumes with high efficiency. This review article discusses the use of LIF detection for the analysis of peptides and proteins in CE. The most common laser sources, basic instrumentation, derivatization modes and set-ups are briefly presented and special attention is paid to the different fluorogenic agents used for pre-, on- and postcapillary derivatization of the functional groups of these compounds. A table summarizing major applications of these derivatization reactions to the analysis of peptides and proteins in CE-LIF and a bibliography with 184 references are provided which covers papers published to the end of 2005.
Sample stacking is a general term for methods in CE which are used for on-line concentration of diluted analytes. During the stacking process, analytes present at low concentrations in a long injected sample zone are concentrated into a short zone (stack). The stacked analytes are then separated and individual zones are detected. Thus stacking provides better separation efficiency and detection sensitivity. Many papers have been published on stacking till now, various procedures have been described, and, many names have been proposed for stacking procedures utilizing the same principles. This contribution brings an easy and unified view on stacking, describes the basic principles utilized, makes a list of recognized operational principles and brings an overview of principal current procedures. Further, it surveys selected recent practical applications ordered according to their operational principles and includes the terms, nicknames, and acronyms used for these actual stacking procedures. This contribution may help both newcomers and experts in the field of CE to orient themselves in the already quite complex topic of sample stacking.