DNA Diagnostics by Capillary Electrophoresis

Klepárnı́k, Karel; Boček, Petr

doi:10.1021/cr0101860

Cited by 73 publications

(68 citation statements)

References 738 publications

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“…CE and CGE are among the most widely accepted separation techniques for the analysis of biologically important polymers, such as nucleic acids, proteins, and complex carbohydrates [1][2][3][4]. Albeit the separation mechanisms for DNA and proteins have been thoroughly investigated in the past decades, similar theoretical treatments for the electrophoretic migration of sugar oligomers in narrow bore capillaries have just been started [5].…”

Section: Introductionmentioning

confidence: 99%

On the electromigration of charged fluorophore‐labeled oligosaccharides in polyethylene oxide solutions

2016

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The separation mechanism of charged fluorophore (aminopyrenetrisulfonate)-labeled maltooligosaccharides with α1-4 linkages was studied in polyethylene oxide (PEO) solutions (MW 300 000 Da) with special interest to possible analyte and/or network deformations as well as potential solute-matrix interactions. The electrophoretic mobilities of the 8-aminopyrene-1,3,6-trisulfonate-labeled maltooligosaccharides were found proportional with their MW(-2/3) . The Arrhenius function was used to determine the activation energy needed by the labeled sugars to migrate through the separation media. With increasing solute size, the activation energy (Ea ) values decreased in polymer concentrations above the entanglement threshold of the PEO, while showed apparently independent function at the entanglement threshold. The observed phenomenon was considered as a result of solute-matrix interaction, which could be alleviated by the addition of an organic modifier to the BGE.

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Section: Introductionmentioning

confidence: 99%

On the electromigration of charged fluorophore‐labeled oligosaccharides in polyethylene oxide solutions

2016

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“…Estimating the size of DNA is critical in genetic analysis associated with human identification [1], species identification [2,3], detecting personal biomarkers [4], analyzing food [5], and categorizing pathogenicity [6]. Moreover, following sample preparation, accurate determination of the size of DNA fragments is a mandatory analytical step in sophisticated high-throughput sequencing techniques.…”

Section: Introductionmentioning

confidence: 99%

Reversible phospholipid nanogels for deoxyribonucleic acid fragment size determinations up to 1500 base pairs and integrated sample stacking

Durney

Bachert

Sloane

et al. 2015

Analytica Chimica Acta

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Phospholipid additives are a cost-effective medium to separate deoxyribonucleic acid (DNA) fragments and possess a thermally-responsive viscosity. This provides a mechanism to easily create and replace a highly viscous nanogel in a narrow bore capillary with only a 10°C change in temperature. Preparations composed of dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC) self-assemble, forming structures such as nanodisks and wormlike micelles. Factors that influence the morphology of a particular DMPC-DHPC preparation include the concentration of lipid in solution, the temperature, and the ratio of DMPC and DHPC. It has previously been established that an aqueous solution containing 10% phospholipid with a ratio of [DMPC]/[DHPC]=2.5 separates DNA fragments with nearly single base resolution for DNA fragments up to 500 base pairs in length, but beyond this size the resolution decreases dramatically. A new DMPC-DHPC medium is developed to effectively separate and size DNA fragments up to 1500 base pairs by decreasing the total lipid concentration to 2.5%. A 2.5% phospholipid nanogel generates a resolution of 1% of the DNA fragment size up to 1500 base pairs. This increase in the upper size limit is accomplished using commercially available phospholipids at an even lower material cost than is achieved with the 10% preparation. The separation additive is used to evaluate size markers ranging between 200 and 1500 base pairs in order to distinguish invasive strains of Streptococcus pyogenes and Aspergillus species by harnessing differences in gene sequences of collagen-like proteins in these organisms. For the first time, a reversible stacking gel is integrated in a capillary sieving separation by utilizing the thermally-responsive viscosity of these self-assembled phospholipid preparations. A discontinuous matrix is created that is composed of a cartridge of highly viscous phospholipid assimilated into a separation matrix of low viscosity. DNA sample stacking is facilitated with longer injection times without sacrificing separation efficiency.

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“…Meanwhile, mutations outside codons 12 and 13 could be also revealed according to the basic principle of TGCE, 16 such as a G/A mutation at codon 10 in the stool sample and cancer tissue of patient no. 21, and a C/T mutation at codon 15 and an A/G mutation at codon 32 in the stool sample of Control-4. Although these mutations were disclosed, the specificity of chip-based TGCE was still up to 93%.…”

Section: Discussionmentioning

confidence: 96%

“…20 The efficiency of detecting genetic variants can be much improved when the electrophoretic process is transplanted onto the microfluidic chip, a new microanalysis system with notable advantages involving higher sensitivity, increased speed, and reduced sample/reagent consumption. 21,22 Buch et al 23 first fabricated a microfluidic device, employing TGCE to rapidly achieve the successful detection of model single-nucleotide polymorphism (SNP) samples. However, the large external heater assemblies in their spatial format increased difficulty with respect to accuracy control and miniaturization.…”

mentioning

confidence: 99%

Rapid detection of low-abundance K-ras mutation in stools of colorectal cancer patients using chip-based temperature gradient capillary electrophoresis

Zhang

Wang

et al. 2011

Laboratory Investigation

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Mutant K-ras provides an independent negative predictive marker for epidermal growth factor receptor (EGFR)-targeted therapy in colorectal cancers (CRCs). Rapid, sensitive, and cost-effective screening for K-ras status will overarch rational personalized medicine. Stool-based DNA testing offers unique advantages for CRC screening such as noninvasiveness, high specificity, and patient compliance, whereas complicated procedures and the low sensitivity of the present approaches have hampered its application on a wide scale. In this study, a chip-based temperature gradient capillary electrophoresis (TGCE) technique was applied to detect low-abundance K-ras mutations under a pooled experiment and analyze K-ras mutations in 30 paired stool samples and cancer tissues of CRC patients and 15 stool samples of healthy volunteers. The chip-based TGCE results showed that the successful analysis of K-ras status could be achieved within 6 min with an extremely low sample consumption of 14 nl. Detection is sensitive enough to reliably report 0.2% mutant CRC cells in a wild-type background, and 0.5 ng of template DNA was sufficient for chip-based TGCE. Of the 30 stool samples of CRC patients analyzed, 17 (57%) harbored K-ras mutations, and the lowest percentage of the detectable mutant K-ras in stool samples was 2%. The coincidence rate for K-ras mutations between stools and tissues obtained by the chip-based method reached 97% (29/30). One of the 15 stool samples of normal controls carried K-ras mutations, producing a specificity of 93%. Clone sequencing data entirely confirmed the results obtained by chip-based TGCE. The study demonstrates that chip-based TGCE is capable of rapidly screening low-abundance K-ras mutations with high sensitivity, reproducibility, simplicity, and significant savings of time and sample. Application of this method to genotype the K-ras gene in stools would provide a potential means for predicting the effectiveness of EGFR-targeted therapy in CRC patients using noninvasive approaches.

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DNA Diagnostics by Capillary Electrophoresis

Cited by 73 publications

References 738 publications

On the electromigration of charged fluorophore‐labeled oligosaccharides in polyethylene oxide solutions

On the electromigration of charged fluorophore‐labeled oligosaccharides in polyethylene oxide solutions

Reversible phospholipid nanogels for deoxyribonucleic acid fragment size determinations up to 1500 base pairs and integrated sample stacking

Rapid detection of low-abundance K-ras mutation in stools of colorectal cancer patients using chip-based temperature gradient capillary electrophoresis

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