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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...

PCR

PCR

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Related Experiment Video

Updated: Jun 9, 2025

Uracil-DNA Glycosylase Assay by Matrix-assisted Laser Desorption/Ionization Time-of-flight Mass Spectrometry Analysis

Uracil-DNA Glycosylase Assay by Matrix-assisted Laser Desorption/Ionization Time-of-flight Mass Spectrometry Analysis

Published on: April 22, 2022

Uracil base PCR implemented for reliable DNA walking.

Rongrong Wang¹, Yinwei Gu¹, Hong Chen¹

¹State Key Laboratory of Food Science and Resource, Nanchang University, Nanchang, 330047, China; International Institute of Food Innovation Co., Ltd., Nanchang University, Nanchang, 330020, China; Sino-German Joint Research Institute, Nanchang University, Nanchang, 330047, China.

Analytical Biochemistry

|October 23, 2024

Summary

Uracil base PCR (UB-PCR) offers a highly specific method for DNA walking to capture unknown genomic sequences. This technique effectively isolates target DNA by selectively amplifying and enriching desired flanking regions.

Keywords:

DNA walking Nested PCR Random annealing Uracil base Uracil-DNA glycosylase Walking primer

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Last Updated: Jun 9, 2025

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Area of Science:

Molecular Biology
Genomics

Background:

Polymerase chain reaction (PCR)-based DNA walking is crucial for identifying unknown flanking genomic sequences.
Existing methods may lack the desired specificity for efficient DNA walking.

Purpose of the Study:

To develop and validate a novel, highly specific PCR-based DNA walking method.
To introduce Uracil base PCR (UB-PCR) as an effective tool for genomic sequence capture.

Main Methods:

Uracil base PCR (UB-PCR) involves replacing thymine with uracil in the initial PCR product (U-DNAs).
A single-primer, single-cycle amplification is performed on U-DNAs, followed by uracil-DNA glycosylase treatment to eliminate U-DNAs.
Secondary and tertiary PCR amplifications are used for exclusive enrichment of the target product.

Main Results:

UB-PCR demonstrated high specificity in capturing unknown flanking genomic sequences.
The method successfully identified unknown sequences bordering three selected genetic sites.
The selective amplification and enrichment strategy proved effective.

Conclusions:

Uracil base PCR (UB-PCR) is a feasible and specific technique for DNA walking.
This method provides an efficient approach for obtaining unknown flanking genomic sequences.
UB-PCR enhances the capability of identifying novel genomic regions.