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Bridging PCR: An Efficient and Reliable Scheme Implemented for Genome-Walking
Zhiyu Lin1,2,3, Cheng Wei2,3, Jinfeng Pei2,3
1School of Chemistry and Chemical Engineering, Nanchang University, Nanchang 330031, China.
Current Issues in Molecular Biology
|January 20, 2023
Summary
Bridging PCR is a novel genome-walking method that overcomes limitations of existing techniques. This approach enhances specificity and efficiency for DNA sequence analysis.
Area of Science:
- Molecular Biology
- Genomics
- Biotechnology
Background:
- Existing genome-walking methods suffer from low specificity and high background noise.
- Complex protocols and composite operations limit the efficiency of current techniques.
Purpose of the Study:
- To develop an efficient and specific genome-walking approach.
- To introduce bridging PCR as an improved method for DNA sequence determination.
Main Methods:
- Bridging PCR utilizes three primers: inner walker primer (IWP), bridging primer (BP), and outer walker primer (OWP).
- The bridging primer (BP) is engineered by joining the outer walker primer (OWP) to the 5'-part of the inner walker primer (IWP).
- A three-step amplification process involving sequential reactions with IWP, BP/OWP, and OWP, paired with nested sequence-specific primers (SSPs), forms the bridging PCR set.
Main Results:
- Non-target products from IWP are end-lengthened by BP, forming hairpin structures that prevent amplification.
- Non-target products initiated by SSP or SSP/IWP are eliminated through nested SSP reactions.
- Only target DNA sequences are accumulated, demonstrating high specificity.
Conclusions:
- Bridging PCR significantly enhances specificity and reduces background in genome walking.
- The method was successfully validated by walking the gadA/R genes in Levilactobacillus brevis and the hyg gene in rice.
