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

Schmidt, Ulrike; Lutz-Bonengel, Sabine; Weisser, Hans-Joachim; Sänger, Timo; Pollak, Stefan; Schön, Ulrike; Zacher, Thomas; Mann, Wolfgang

doi:10.1007/s00414-005-0041-2

Cited by 36 publications

(32 citation statements)

References 33 publications

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“…Investigations of low volume multiplex reactions on a chemically structured chip showed successful amplification from as little as 32 pg of cell line DNA revealing a full STR profile [22]. However, some problems occurred when working with low volume on-chip PCR: dropout of loci and single alleles were observed, which can be interpreted as stochastic effects due to pipetting of very small volumes [14].…”

Section: Introductionmentioning

confidence: 99%

Low volume amplification and sequencing of mitochondrial DNA on a chemically structured chip

et al. 2006

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Low volume (LV) amplification (1 μL) of nuclear DNA (nucDNA) on a chemically structured chip is an appropriate and highly sensitive method to simultaneously amplify amelogenin and 15 forensically relevant short tandem repeats (STR). In this study, a combined method using on-chip LV amplification of mitochondrial DNA (mtDNA) and subsequent on-chip LV cycle sequencing was established to obtain a method, which is sensitive and robust enough to allow reliable analysis of DNA amounts representing the single cell level. All the necessary steps of the procedure-except for the purification of the sequencing products-were accomplished within the same final 2-μL reaction volume.

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Section: Introductionmentioning

confidence: 99%

Low volume amplification and sequencing of mitochondrial DNA on a chemically structured chip

et al. 2006

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“…2). The method of minisequencing itself is ideally suited for the chemically structured chip as typing results are unambiguous; the difficulties typically observed with STR analysis do not occur with single-nucleotide extension [10]. However, batch-specific differences in the slide quality have been observed.…”

Section: Resultsmentioning

confidence: 97%

“…Additionally, the method should be highly sensitive to enable multiple typing of various markers from limited amounts of sample. As described previously, an enhancement of polymerase chain reaction (PCR) sensitivity can be achieved by the reduction of the total PCR reaction volume [10,11]. A low-volume (LV) PCR performed in a 1-µL reaction on the surface of a chemically structured glass slide also presents enormous cost efficiency due to the reduction of PCR reagents [10].…”

Section: Introductionmentioning

confidence: 99%

Reduced-volume and low-volume typing of Y-chromosomal SNPs to obtain Finnish Y-chromosomal compound haplotypes

et al. 2009

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Single-nucleotide extension is a widespread method for typing Y-chromosomal single-nucleotide polymorphisms. In our study, we validated a multiplex minisequencing assay in a reduced-volume and in a low-volume approach. A four-plex assay was performed in a 6-microL multiplex reaction in 96-well microtiter reaction plates, which can be directly used for capillary electrophoresis. In a second approach, a six-plex assay was performed on a chemically structured glass slide. Both techniques have proven to be highly sensitive as well as time- and cost-saving, which makes them a valuable option not only for forensic purposes but also for population genetic studies where large sample numbers have to be analyzed. In the present paper, both techniques are compared and applied to analyze a population sample from the area of Turku, Finland. The most common haplogroup was found to be N1c*, which is nearly absent in western and central European populations. Additionally, 11 short tandem repeat markers were analyzed to further discriminate Y-chromosomal lineages.

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“…Compared to PCR analysis in larger reaction volumes, this leads to an increased amplification sensitivity and efficiency of PCR which is necessary for minute amounts of DNA [25,26] . Application of fluorescent PCR, which is standard in PGD nowadays, as well as the optimization of PCR conditions and nested PCR protocols are further strategies for additionally increasing amplification efficiency and detection sensitivity [1,27] .…”

Section: Discussionmentioning

confidence: 99%

Single Cell Analysis of Mutations in the <i>APC</i> Gene

et al. 2009

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Introduction: Mutation analysis of inherited monogenic diseases is an important aspect of preimplantation genetic diagnosis. Familial adenomatous polyposis of the colon is an autosomal dominant inherited disorder caused by mutations in the tumor suppressor gene adenomatous polyposis coli (APC). A characteristic of this severe disease is the development of thousands of polyps in the colon which untreated evolve into malignant colon carcinomas. Here we present a novel approach for the establishment of mutation detection in the APC gene in single cells applicable for preimplantation genetic diagnosis. Methods: Single fixed lymphocytes were isolated via laser microdissection and transferred to reaction centers of planar chemically structured glass slides via a recently developed horizontal low-pressure single particle adsorbing transfer system (SPATS). A multiplex nested polymerase chain reaction protocol in a 1-μl reaction volume, followed by sequencing and fragment length analysis was applied in order to detect mutations in the APC gene. Results: Reliable isolation and transfer of single lymphocytes was demonstrated. High amplification efficiency and low allelic drop out (ADO) rates for polymorphic microsatellite markers and mutation specific amplification products of various mutations in the APC gene were obtained from fixed single cells. Conclusions: This novel nanotechnological downscaling approach enables a reliable validation of genetic testing using diploid single lymphocytes, and will open a wide range of single cell diagnostics.

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

Cited by 36 publications

References 33 publications

Low volume amplification and sequencing of mitochondrial DNA on a chemically structured chip

Low volume amplification and sequencing of mitochondrial DNA on a chemically structured chip

Reduced-volume and low-volume typing of Y-chromosomal SNPs to obtain Finnish Y-chromosomal compound haplotypes

Single Cell Analysis of Mutations in the <i>APC</i> Gene

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