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Southern Blot

Southern Blot

Agarose gel electrophoresis is very useful in separating DNA fragments by size. Running a DNA ladder containing fragments of the known length alongside the sample helps determine the approximate length of the sample DNA fragments. However, additional steps are needed to verify the sequence identity of the sample DNA fragments.
Denatured DNA fragments must be transferred onto a carrier membrane from the gel to make it accessible to a probe - a small ssDNA fragment complementary to the target DNA...

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Quantification of ssDNA Scaffold Production by Ion-Pair Reverse Phase Chromatography.

A Rita Silva-Santos^1,2, Sara Sousa Rosa^1,2, Marco P C Marques³

¹iBB-Institute for Bioengineering and Biosciences, Department of Bioengineering, Instituto Superior Técnico, Universidade de Lisboa, Av. Rovisco Pais 1, 1049-001 Lisboa, Portugal.

|June 3, 2024

Summary

This study introduces a new HPLC method for accurately quantifying DNA scaffolds used in DNA origami. The method improves the production of single-stranded DNA (ssDNA) for nanotechnology applications.

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Area of Science:

Nanotechnology
Molecular Biology
Biochemistry

Background:

DNA origami utilizes DNA nanostructures for applications like drug delivery and biosensors.
Production of DNA scaffolds typically involves asymmetric PCR or bacterial infection, with quantification challenges.
Existing quantification methods (gel electrophoresis, UV absorbance) lack accuracy due to impurities.

Purpose of the Study:

To develop a precise method for quantifying single-stranded DNA (ssDNA) scaffolds.
To improve the optimization of ssDNA production methods.
To enable accurate quantification of ssDNA from various production routes.

Main Methods:

Ion-pair reversed-phase (IP-RP) high-performance liquid chromatography (HPLC) was employed.
ssDNA scaffolds produced via asymmetric PCR were separated and quantified.
ssDNA from phage infection was quantified using M13mp18 ssDNA as a standard.

Main Results:

IP-RP chromatography effectively separated ssDNA from impurities and double-stranded DNA (dsDNA) byproducts.
The method provided accurate quantification of both ssDNA and dsDNA.
Optimization of asymmetric PCR for ssDNA production was guided by the new quantification method.
ssDNA from phage infection was successfully quantified.

Conclusions:

IP-RP HPLC offers a sensitive and selective method for DNA scaffold quantification.
This technique enhances the optimization of ssDNA production for DNA origami.
The method provides accurate measurements crucial for advancing nanotechnology applications.