Low-volume amplification on chemically structured chips using the PowerPlex16 DNA amplification kit

University of Freiburg, Freiburg, Baden-Württemberg, Germany
Deutsche Zeitschrift für die Gesamte Gerichtliche Medizin (Impact Factor: 2.6). 02/2006; 120(1):42-8. DOI: 10.1007/s00414-005-0041-2
Source: PubMed

ABSTRACT In forensic DNA analysis, improvement of DNA typing technologies has always been an issue. It has been shown that DNA amplification in low volumes is a suitable way to enhance the sensitivity and efficiency of amplification. In this study, DNA amplification was performed on a flat, chemically structured glass slide in 1-microl reaction volumes from cell line DNA contents between 1,000 and 4 pg. On-chip DNA amplification reproducibly yielded full allelic profiles from as little as 32 pg of template DNA. Applicability on the simultaneous amplification of 15 short tandem repeats and of a segment of the Amelogenin gene, which are routinely used in forensic DNA analysis, is shown. The results are compared to conventional in-tube amplification carried out in 25-microl reaction volumes.

1 Follower
  • [Show abstract] [Hide abstract]
    ABSTRACT: Nucleic acid analysis in a single cell is very important, but the extremely small amount of template in a single cell requires a detection method more sensitive than the conventional method. In this paper, we describe a novel assay allowing a single cell genotyping by coupling improved linear-after-the-exponential-PCR (imLATE-PCR) on a modified glass slide with highly sensitive pyrosequencing. Due to the significantly increased yield of ssDNA in imLATE-PCR amplicons, it is possible to employ pyrosequencing to sequence the products from 1 μL chip PCR which directly used a single cell as the starting material. As a proof-of-concept, the 1555A>G mutation (related to inherited deafness) on mitochondrial DNA and the SNP 2731C>T of the BRCA1 gene on genomic DNA from a single cell were successfully detected, indicating that our single-cell-pyrosequencing method has high sensitivity, simple operation and is low cost. The approach has promise to be of efficient usage in the fields of diagnosis of genetic disease from a single cell, for example, preimplantation genetic diagnosis (PGD).
    The Analyst 07/2013; 138(17). DOI:10.1039/c3an00821e · 3.91 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: We developed and characterized a novel picoliter droplet-in-oil array generated by a double-inkjet printing method on a uniform hydrophobic silicon chip, specifically designed for quantitative polymerase chain reaction (qPCR) analysis. Double-inkjet printing was proposed to efficiently address the evaporation issues of picoliter droplets during array generation on a planar substrate without the assistance of a humidifier or glycerol. The method utilizes piezoelectric inkjet printing equipment to precisely eject a reagent droplet into an oil droplet, which had first been dispensed on a hydrophobic and oleophobic substrate. No evaporation, random movement, or cross-contamination was observed during array fabrication and thermal cycling. We demonstrated the feasibility and effectiveness of this novel double-inkjet method for real-time PCR analysis. This method can readily produce multivolume droplet-in-oil arrays with volume variations ranging from picoliters to nanoliters. This feature would be useful for simultaneous multivolume PCR experiments aimed at wide and tunable dynamic ranges. These double-inkjet-based picoliter droplet arrays may have potential for multiplexed applications that require isolated containers for single cell cultures, single molecular enzymatic assays, or digital PCR, and provide an alternative option for generating droplet arrays on planar substrates without chemical patterning.
    Lab on a Chip 07/2014; 14(18). DOI:10.1039/C4LC00598H · 5.75 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Functionalities of microchip-based analytical systems for nucleic acids analysis are highlighted and driving forces for the future progress are reviewed. The review shows the main advantages of microchip-based systems and discusses up-to-date achievements in this area. Among the main advantages are a high speed of analysis, high rates of heating and cooling, a low reagents consumption, and a high potential to integrate several analytical processes in a single microchip device.
    Russian Journal of General Chemistry 12/2013; 82(12). DOI:10.1134/S1070363212120353 · 0.42 Impact Factor