A High-Throughput Protocol for Isolating Cell-Free Circulating Tumor DNA from Peripheral Blood

Pandoh, Pawan; Corbett, Richard; McDonald, Helen; Alcaide, Miguel; Kirk, Heather; Trinh, Eva; Haile, Simon; MacLeod, Tina; Smailus, Duane E.; Bilobram, Steve; Mungall, Andrew J.; Ma, Yussanne; Moore, Richard A.; Coope, Robin; Zhao, Yongjun; Jones, Steven J.M.; Holt, Robert A.; Karsan, Aly; Morin, Ryan D.; Marra, Marco A.

doi:10.2144/btn-2018-0148

Cited by 13 publications

(6 citation statements)

References 21 publications

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“…The absence of a centrifugation step allows to automate the process and reduce the risk of cross-contamination [23]. It allows one to isolate a product from large volumes with a low DNA concentration without increasing the process execution time [24,25]. Size fractionation of DNA molecules is an important advantage that has found application in the preparation of samples for whole genome sequencing.…”

Section: Discussionmentioning

confidence: 99%

Optimization of PCR Purification Using Silica-Coated Magnetic Beads

Berdimuratova¹,

Amirgazin²,

Kuibagarov³

et al. 2020

Eurasian Journal of Applied Biotechnology

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Purification of nucleic acids is still an important step in molecular genetic research. The development of whole genome sequencing technologies has increased the requirements for the purity of the nucleic acids used, and also required the selection of DNA fragments by size. Buffer systems that contain PEG/NaCl solutions and silica-coated magnetic beads allow to purify nucleic acids and selectively sorb certain sizes of DNA. In this article, we present a simple protocol for the purification of PCR products with the ability to absorb the required DNA molecules. It was determined that the use of an optimized PEG / NaCl buffer system with magnetic silica gel in a ratio of 1.5: 1 with a PCR product allows to get rid of DNA fragments 100 and less base pairs (bp), as well as other contaminants, while maintaining this is more than 90% of the DNA in solution. The ratio of 0.35: 1 allows for high-affinity sorption of DNA molecules larger than 400 bp. The practical use of the obtained data allows us to improve the quality of sequencing without increasing the cost of research.

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Section: Discussionmentioning

confidence: 99%

Optimization of PCR Purification Using Silica-Coated Magnetic Beads

Berdimuratova¹,

Amirgazin²,

Kuibagarov³

et al. 2020

Eurasian Journal of Applied Biotechnology

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“…In recent years, several companies developed a range of DNA isolation kits of which only the "dedicated" kits were recommended for the extraction of cfDNA [192]. In several papers, a variety of different kits were compared [84,85,93,[193][194][195][196][197][198][199][200][201][202][203][204]. From these results, one can conclude that bead-based isolation methods worked somewhat better than membrane-based kits and that the former ones are especially useful for the isolation of low-molecularweight DNA.…”

Section: Dna Isolationmentioning

confidence: 99%

Pre-analytical issues in liquid biopsy – where do we stand?

Fleischhacker

Schmidt

2020

Journal of Laboratory Medicine

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It is well documented that in the chain from sample to the result in a clinical laboratory, the pre-analytical phase is the weakest and most vulnerable link. This also holds for the use and analysis of extracellular nucleic acids. In this short review, we will summarize and critically evaluate the most important steps of the pre-analytical phase, i.e. the choice of the best control population for the patients to be analyzed, the actual blood draw, the choice of tubes for blood drawing, the impact of delayed processing of blood samples, the best method for getting rid of cells and debris, the choice of matrix, i.e. plasma vs. serum vs. other body fluids, and the impact of long-term storage of cell-free liquids on the outcome. Even if the analysis of cell-free nucleic acids has already become a routine application in the area of non-invasive prenatal screening (NIPS) and in the care of cancer patients (search for resistance mutations in the EGFR gene), there are still many unresolved issues of the pre-analytical phase which need to be urgently tackled.

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“…Relatively low ctDNA levels in standard blood samples and background cfDNA unrelated to tumor cells make detection a challenge. Therefore, the standard procedure for blood collection and DNA isolation should be finalized prior to analysis 22,23…”

Section: Origin and Detection Of Cfdna/ctdnamentioning

confidence: 99%

<p>Circulating Cell-Free DNA or Circulating Tumor DNA in the Management of Ovarian and Endometrial Cancer</p>

Chen¹,

Zhang²,

Wang³

et al. 2019

OTT

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Ovarian cancer (OC) is the most lethal cancer of all gynecological malignancies, while endometrial cancer (EC) is the most common one. Current strategies for OC/EC diagnosis consist of the extraction of a solid tissue from the affected area. This sample enables the study of specific biomarkers and the genetic nature of the tumor. However, the tissue extraction is risky and painful for the patient and in some cases is unavailable in inaccessible tumors. Moreover, a tissue biopsy is expensive and requires a highly skilled gynecological surgery to pinpoint accurately which cannot be applied repeatedly. New alternatives that overcome these drawbacks are rising up nowadays, such as liquid biopsy. A liquid biopsy is the analysis of biomarkers in a non-solid biological tissue, mainly blood, which has remarkable advantages over the traditional method. The most studied cancer non-invasive biomarkers are circulating tumor cells (CTCs), circulating tumor DNA (ctDNA), and circulating free DNA (cfDNA). These circulating biomarkers play a key role in the understanding of metastasis and tumorigenesis, which could provide a better insight into the evolution of the tumor dynamics during treatment and disease progression. Liquid biopsy is an emerging non-invasive, safe and effective method with considerable potential for clinical diagnosis and treatment management in patients with OC and EC. Analysis of cfDNA and ctDNA will provide a better characterization of biomarkers and give rise to a wide range of clinical applications, such as early detection of OC/EC, the prediction of treatment responses due to the discovery of personalized tumor-related biomarkers, and therapeutic response monitoring.

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A High-Throughput Protocol for Isolating Cell-Free Circulating Tumor DNA from Peripheral Blood

Cited by 13 publications

References 21 publications

Optimization of PCR Purification Using Silica-Coated Magnetic Beads

Optimization of PCR Purification Using Silica-Coated Magnetic Beads

Pre-analytical issues in liquid biopsy – where do we stand?

<p>Circulating Cell-Free DNA or Circulating Tumor DNA in the Management of Ovarian and Endometrial Cancer</p>

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