Single-cell PCR of genomic DNA enabled by automated single-cell printing for cell isolation

Stumpf, Fabian; Schoendube, Jonas; Gross, André; Rath, Christin; Niekrawietz, S.; Koltay, Peter; Roth, Günter

doi:10.1016/j.bios.2015.03.008

Cited by 47 publications

(29 citation statements)

References 27 publications

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“…While the recommended amount of template DNA, according to the manufacturer (Available online: ), is 10 4 copies (~34.5 ng of human genomic DNA) or above, evaluation using the PCR protocol described in this study achieved a limit of detection (LOD) of ≤1.25 ng of human genomic DNA (Methods S2). Further optimizations, including increasing the number of PCR cycles [35], may bring the LOD down to the theoretical limit of a single genomic copy [36]; nevertheless, as a clinical laboratory performing diagnostic testing, it may be preferable to balance among economical use of template DNA, reasonable number of PCR cycles and standardizing laboratory protocols to avoid excessive pipetting and manual dilution steps. With regard to these, PCR-HRM is considered less robust compared to Sanger sequencing and PCR-RFLP, which depend only on the presence of adequate amplicon for subsequent cycle sequencing and restriction enzyme digestion, rather than comparable amplification efficiency within a critical number of cycles [37].…”

Section: Discussionmentioning

confidence: 99%

Comparison of Direct Sequencing, Real-Time PCR-High Resolution Melt (PCR-HRM) and PCR-Restriction Fragment Length Polymorphism (PCR-RFLP) Analysis for Genotyping of Common Thiopurine Intolerant Variant Alleles NUDT15 c.415C>T and TPMT c.719A>G (TPMT*3C)

Fong

Poon

2017

Diagnostics

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Thiopurine intolerance and treatment-related toxicity, such as fatal myelosuppression, is related to non-function genetic variants encoding thiopurine S-methyltransferase (TPMT) and Nudix hydrolase 15 (NUDT15). Genetic testing of the common variants NUDT15:NM_018283.2:c.415C>T (Arg139Cys, dbSNP rs116855232 T allele) and TPMT: NM_000367.4:c.719A>G (TPMT*3C, dbSNP rs1142345 G allele) in East Asians including Chinese can potentially prevent treatment-related complications. Two complementary genotyping approaches, real-time PCR-high resolution melt (PCR-HRM) and PCR-restriction fragment length morphism (PCR-RFLP) analysis were evaluated using conventional PCR and Sanger sequencing genotyping as the gold standard. Sixty patient samples were tested, revealing seven patients (11.7%) heterozygous for NUDT15 c.415C>T, one patient homozygous for the variant and one patient heterozygous for the TPMT*3C non-function allele. No patient was found to harbor both variants. In total, nine out of 60 (15%) patients tested had genotypic evidence of thiopurine intolerance, which may require dosage adjustment or alternative medication should they be started on azathioprine, mercaptopurine or thioguanine. The two newly developed assays were more efficient and showed complete concordance (60/60, 100%) compared to the Sanger sequencing results. Accurate and cost-effective genotyping assays by real-time PCR-HRM and PCR-RFLP for NUDT15 c.415C>T and TPMT*3C were successfully developed. Further studies may establish their roles in genotype-informed clinical decision-making in the prevention of morbidity and mortality due to thiopurine intolerance.

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Section: Discussionmentioning

confidence: 99%

Comparison of Direct Sequencing, Real-Time PCR-High Resolution Melt (PCR-HRM) and PCR-Restriction Fragment Length Polymorphism (PCR-RFLP) Analysis for Genotyping of Common Thiopurine Intolerant Variant Alleles NUDT15 c.415C>T and TPMT c.719A>G (TPMT*3C)

Fong

Poon

2017

Diagnostics

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“…Instead, as described in previous sections, combinations of both materials can deliver more complex building blocks for constructs produced using additive manufacturing. This is an important consideration, as currently available additive manufacturing tools for single-cell printing do not reach further than printing of 2D patterns, despite efforts placed in improving nozzle designs and positioning stages [199,200], although such 2D patterns have proven to be useful for single-cell genomic analysis [199], among others [201,202]. In this context, microfluidic solutions may be advantageous for separation and isolation of cells ahead of being expelled through the nozzle orifice.…”

Section: Building Blocks For Bioprintingmentioning

confidence: 99%

High-throughput screening approaches and combinatorial development of biomaterials using microfluidics

2016

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Microfluidics, being a technology characterized by the engineered manipulation of fluids at the submillimeter scale, offers some interesting tools that can advance biomedical research and development. Screening platforms based on microfluidic technologies that allow high-throughput and combinatorial screening may lead to breakthrough discoveries not only in basic research but also relevant to clinical application. This is further strengthened by the fact that reliability of such screens may improve, since microfluidic systems allow close mimicking of physiological conditions. Finally, microfluidic systems are also very promising as micro factories of a new generation of natural or synthetic biomaterials and constructs, with finely controlled properties.

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“…Thermal, single cell lysis for single cell PCR has been executed off chip using a standard thermocycler and PCR tubes 192 . The lysis step required a 95° C hold for 90 seconds.…”

Section: Sample Preparationmentioning

confidence: 99%

Microfluidic Sample Preparation for Single Cell Analysis

2015

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Single-cell PCR of genomic DNA enabled by automated single-cell printing for cell isolation

Cited by 47 publications

References 27 publications

Comparison of Direct Sequencing, Real-Time PCR-High Resolution Melt (PCR-HRM) and PCR-Restriction Fragment Length Polymorphism (PCR-RFLP) Analysis for Genotyping of Common Thiopurine Intolerant Variant Alleles NUDT15 c.415C>T and TPMT c.719A>G (TPMT*3C)

Comparison of Direct Sequencing, Real-Time PCR-High Resolution Melt (PCR-HRM) and PCR-Restriction Fragment Length Polymorphism (PCR-RFLP) Analysis for Genotyping of Common Thiopurine Intolerant Variant Alleles NUDT15 c.415C>T and TPMT c.719A>G (TPMT*3C)

High-throughput screening approaches and combinatorial development of biomaterials using microfluidics

Microfluidic Sample Preparation for Single Cell Analysis

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