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Bridging PCR-Based Genome-Walking Protocol.

Mou Li^1,2,3, Yinwei Gu^1,2,3, Qingchun Tang^1,2,3

¹State Key Laboratory of Food Science and Resources, Nanchang University, Nanchang, China.

|December 12, 2025

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This study introduces a novel genome walking method using bridging PCR to eliminate non-target DNA amplification. The technique enhances specificity and simplifies experimental operation for amplifying unknown DNA regions.

Area of Science:

Molecular Biology
Genomics

Background:

Genome walking is crucial for amplifying unknown DNA flanking known sequences.
Current genome walking protocols face challenges in operational simplicity and specificity.
Non-target product amplification contributes significantly to background noise.

Purpose of the Study:

To introduce a novel genome walking protocol based on bridging PCR.
To enhance the specificity and reduce background noise in genome walking.
To provide a universal and operationally simpler genome walking method.

Main Methods:

A novel bridging PCR protocol is proposed, utilizing a bridging primer.
The bridging primer features an oligomer tail attached to the 5' end of the walker primer's 5' region.
Non-target DNA is lengthened at both ends by the bridging primer, promoting intrastrand annealing.

Keywords:

Bridging PCR Bridging primer Genome walking Intrastrand annealing Inverted repeat ends of DNA Sequence-specific primer Walker primer

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Main Results:

The novel protocol effectively eliminates non-target DNA amplification.
Target DNA amplification remains unaffected by the new method.
The method relies on increased intrastrand annealing of lengthened non-target DNA.

Conclusions:

The bridging PCR method offers a universal solution for specific genome walking.
This technique significantly reduces background noise by eliminating non-target products.
The protocol improves experimental operation and methodological specificity in DNA amplification.