Quantification of Trace-Level DNA by Real-Time Whole Genome Amplification

Kang, Min-Jung; Yu, Hannah; Kim, Sook-Kyung; Park, Sang-Ryoul; Yang, Inchul

doi:10.1371/journal.pone.0028661

Cited by 9 publications

(5 citation statements)

References 21 publications

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“…The DNA–RNA chimeric product was exponentially produced with as little as 300 nM enzyme and 0.2 nM template, except with the 12 + 12 template, which required 2 nM template (Figure B and Figures S4–S7). As observed in other studies, − slight decreases in fluorescence were observed in the qPCT curves at long times, which is likely due to SYBR green I dye instability. Product was confirmed by PAGE (Figure C and Figures S4–S7), and the desired RNA oligonucleotide was easily obtained by incubation with TurboDNase (Figures F and S9).…”

Section: Resultssupporting

confidence: 82%

Polymerase Chain Transcription: Exponential Synthesis of RNA and Modified RNA

Chen

Romesberg

2017

J. Am. Chem. Soc.

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There is increasing demand for RNA and modified RNA oligonucleotides, but in contrast to DNA oligonucleotides, they are typically prohibitively expensive to chemically synthesize, and unlike longer RNAs, they are only inefficiently produced by in vitro transcription, especially when modified. To address these challenges, we previously reported the evolution of a thermostable DNA polymerase, SFM4-3, that more efficiently accepts substrates with 2'-substituents. We now show that SFM4-3 efficiently transcribes RNA or 2'-F-modified RNA and that it also efficiently PCR amplifies oligonucleotides of mixed RNA and DNA composition. In addition, with thermocycling and the use of a novel DNA template, we demonstrate a polymerase chain transcription (PCT) reaction that results in the exponential production of orders of magnitude more RNA or modified RNA than is available by conventional transcription. PCT is more efficient and general than conventional transcription and can produce large amounts of any RNA or modified RNA oligonucleotide at a fraction of the cost of chemical synthesis.

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Section: Resultssupporting

confidence: 82%

Polymerase Chain Transcription: Exponential Synthesis of RNA and Modified RNA

Chen

Romesberg

2017

J. Am. Chem. Soc.

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“…The contamination level of the host cell nucleic acid content in the purified mAb was determined by qPCR analysis. The assay was based on a previous work (Kang et al 2011 ). Briefly, a standard curve from 5 nanogram to 500 attogram of DNA was generated from a CHO-K1 cell DNA stock (IDT, Coralville, Iowa, USA).…”

Section: Methodsmentioning

confidence: 99%

Effects of process intensification on homogeneity of an IgG1:κ monoclonal antibody during perfusion culture

Liang,

Madhavarao,

Morris

et al. 2024

Appl Microbiol Biotechnol

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The pharmaceutical industry employs various strategies to improve cell productivity. These strategies include process intensification, culture media improvement, clonal selection, media supplementation and genetic engineering of cells. However, improved cell productivity has inherent risk of impacting product quality attributes (PQA). PQAs may affect the products’ efficacy via stability, bioavailability, or in vivo bioactivity. Variations in manufacturing process may introduce heterogeneity in the products by altering the type and extent of N-glycosylation, which is a PQA of therapeutic proteins. We investigated the effect of different cell densities representing increasing process intensification in a perfusion cell culture on the production of an IgG1-κ monoclonal antibody from a CHO-K1 cell line. This antibody is glycosylated both on light chain and heavy chain. Our results showed that the contents of glycosylation of IgG1-κ mAb increased in G0F and fucosylated type glycans as a group, whereas sialylated type glycans decreased, for the mAb whole protein. Overall, significant differences were observed in amounts of G0F, G1F, G0, G2FS1, and G2FS2 type glycans across all process intensification levels. G2FS2 and G2 type N-glycans were predominantly quantifiable from light chain rather than heavy chain. It may be concluded that there is a potential impact to product quality attributes of therapeutic proteins during process intensification via perfusion cell culture that needs to be assessed. Since during perfusion cell culture the product is collected throughout the duration of the process, lot allocation needs careful attention to process parameters, as PQAs are affected by the critical process parameters (CPPs). Key points • Molecular integrity may suffer with increasing process intensity. • Galactosylated and sialylated N-glycans may decrease. • Perfusion culture appears to maintain protein charge structure.

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“…The primers of DOP-PCR are placed at the 3' end, randomly in the middle, and at the 5' end. This method has been successfully demonstrated in the determination of the human placental DNA ranging from 80fg to 8ng [43].…”

Section: Polymerase Chain Reaction (Pcr) Amplificationmentioning

confidence: 99%

Recent Advances in Forensic DNA Analysis

Romeika

Yan²

2014

J Forensic Res

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Forensic DNA (deoxyribonucleic acid) analysis or DNA profiling has played a major role in the criminal justice system. New techniques and technologies for DNA profiling continue to evolve every year. This paper reviews the literature reported during January 2011 through June 2013 in the field of forensic DNA analysis. Recent advances in almost all aspects of DNA analysis-which include sample collection, storage, and pretreatment, DNA extraction, DNA quantitation, quality assurance of DNA testing, and DNA databases are discussed. New developments now enable new kinds of forensically relevant information to be extracted from limited quantities of biological samples, and offer unprecedented potential for high-throughput DNA analysis.

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Quantification of Trace-Level DNA by Real-Time Whole Genome Amplification

Cited by 9 publications

References 21 publications

Polymerase Chain Transcription: Exponential Synthesis of RNA and Modified RNA

Polymerase Chain Transcription: Exponential Synthesis of RNA and Modified RNA

Effects of process intensification on homogeneity of an IgG1:κ monoclonal antibody during perfusion culture

Recent Advances in Forensic DNA Analysis

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