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DNA Isolation

DNA Isolation

DNA from cells is required for many biotechnology and research applications, such as molecular cloning. To remove and purify DNA from cells, researchers use various methods of DNA extraction. While the specifics of different protocols may vary, some general concepts underlie the process of DNA extraction.

Overview Of Cell Separation And Isolation

Overview Of Cell Separation And Isolation

Cell separation was first achieved in 1964 by S. H. Seal, who separated large tumor cells from the smaller blood cells using filtration. Two years later, Pohl and Hawk performed experiments on how cells respond differently to a nonuniform electric field based on the cell type. Such observations were the inception of cell separation methods, which allow isolating a single cell type from a heterogeneous sample.

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A phenol-chloroform free method for cfDNA isolation from cell conditioned media: development, optimization and

Dhruv Das¹, Rao Avssn², Rajani Kant Chittela¹

¹Applied Genomics Section, Bioscience Group, Bhabha Atomic Research Centre, Mumbai, 400085, India; Homi Bhabha National Institute (HBNI), Anushaktinagar, Trombay, Mumbai, 400094, India.

Analytical Biochemistry

|December 29, 2023

Summary

We developed a safe, rapid, and cost-effective method for extracting cell-free DNA (cfDNA) from cell culture media. This new protocol yields cfDNA comparable or superior to existing methods, with no compromise on quality.

Keywords:

Biomarker Cell culture Cell-free DNA Fluorescence-based assay qPCR

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Area of Science:

Biochemistry
Molecular Biology
Genomics

Background:

Cell-free DNA (cfDNA) is increasingly recognized as a valuable biomarker for diagnostics, prognostics, and theranostics.
Understanding cfDNA origins and biological significance is crucial for clinical utility.
In vitro cell culture models offer insights into cfDNA biology, but cfDNA extraction efficiency from cell conditioned media (CCM) varies significantly with methodology.

Purpose of the Study:

To develop and evaluate a novel phenol-chloroform-free cfDNA extraction method from CCM.
To compare the developed method against existing published protocols and commercial kits.
To assess the yield and quality of cfDNA extracted using different methods.

Main Methods:

Development of a phenol-chloroform-free cfDNA extraction protocol.
Comparison with three published cfDNA extraction methods and four commercial kits.
Quantification of extracted cfDNA using Real-Time PCR (qPCR) and a Qubit assay.

Main Results:

The developed method provides cfDNA yields that are higher or comparable to other tested methods.
qPCR quantification showed consistent relative trends across different targets, despite variations in absolute concentration.
The new protocol is safe, rapid, cost-effective, and requires minimal hands-on time.

Conclusions:

A safe, rapid, and cost-effective cfDNA extraction protocol from CCM was successfully developed.
The developed method achieves comparable or superior cfDNA yields without compromising quality.
This protocol offers a practical solution for cfDNA isolation in research settings.