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DNA Isolation

DNA Isolation

DNA isolation protocols can be fast and straightforward or complex and time-consuming depending on the type and quality of DNA required for further processing. For example, plasmid DNA extraction is a bit more complicated than genomic DNA extraction because of the need for an appropriate lysis method to separate plasmid DNA from gDNA during isolation. However, for specific applications, such as long-range DNA sequencing that require a good yield of high- quality DNA samples, we need to follow...

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Mapping Mammalian 3D Genome Interactions with Micro-C-XL

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Published on: November 3, 2023

Efficient removal of DNA from proteomic samples prior to two-dimensional map analysis.

Paolo Antonioli¹, Angela Bachi, Elisa Fasoli

¹Department of Chemistry, Materials and Chemical Engineering Giulio Natta, Politecnico di Milano, Milan, Italy.

Journal of Chromatography. A

|December 17, 2008

Summary

Efficient DNA removal from protein samples is crucial for accurate proteome analysis. A novel two-phase extraction method at alkaline pH effectively eliminates DNA, improving proteome data quality.

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Area of Science:

Biochemistry
Proteomics
Molecular Biology

Background:

Existing DNA removal methods for proteome analysis often involve protein precipitation or DNase treatment.
These conventional techniques are frequently inadequate, leading to artifacts like streaking on 2D gels and loss of proteins.
High DNA levels in samples, particularly from tissues and nuclei, significantly hinder proteome analysis.

Purpose of the Study:

To develop and validate a more efficient method for DNA removal from protein samples.
To improve the quality and comprehensiveness of proteome analysis by minimizing DNA interference.
To investigate the effectiveness of a two-phase extraction method for DNA removal in proteomic workflows.

Main Methods:

A two-phase extraction using chloroform/phenol/isoamyl alcohol was adapted for DNA removal from protein samples.
The extraction was optimized and performed at alkaline pH (≥9.5).
The method was tested on Escherichia coli lysates and purified eukaryotic nuclei extracts.

Main Results:

The alkaline two-phase extraction method demonstrated highly efficient DNA removal.
Two-dimensional gel electrophoresis maps showed a complete absence of vertical and horizontal streaking.
Significantly more protein spots were detected, especially basic proteins, indicating reduced co-precipitation with DNA.

Conclusions:

Alkaline two-phase extraction is a superior method for removing DNA from protein samples prior to proteome analysis.
This method enhances proteome data quality by eliminating artifacts and recovering previously lost basic proteins.
The alkaline pH is hypothesized to disrupt DNA-protein complexes via Coulombic repulsion, facilitating complete DNA removal.