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Wristwatch PCR: A Versatile and Efficient Genome Walking Strategy
Lingqin Wang1,2, Mengya Jia1,2, Zhaoqin Li3
1State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang, China.
Frontiers in Bioengineering and Biotechnology
|May 2, 2022
Summary
We developed wristwatch (WW) PCR, an efficient genome walking technique using novel primers to retrieve unknown flanking DNA sequences. This method successfully identified DNA flanking specific genes in bacteria and plants.
Area of Science:
- Molecular Biology
- Genomics
- Biotechnology
Background:
- Genome walking is crucial for identifying unknown DNA sequences adjacent to known regions.
- Existing genome walking methods can be complex and time-consuming.
Purpose of the Study:
- To introduce and validate wristwatch (WW) PCR, a novel and efficient genome walking technique.
- To demonstrate the utility of WW PCR in accessing flanking DNA regions.
Main Methods:
- Developed WW primers (WWPs) with 5'/3' overlap and a heterologous interval for wristwatch-like structure formation.
- Employed a three-nested PCR approach (primary, secondary, tertiary) using WWPs under varying stringency cycles.
- Utilized gene-specific primers (GSPs) for exponential amplification of target single-stranded DNAs (ssDNAs).
Main Results:
- Successfully validated WW-PCR by identifying unknown flanking DNA regions.
- Demonstrated success in accessing regions flanking the glutamate decarboxylase gene in *Lactobacillus brevis* CD0817.
- Confirmed efficacy in retrieving the hygromycin gene flanking region in rice.
Conclusions:
- WW-PCR is an efficient and effective genome walking technique.
- Offers an attractive alternative to existing methods for retrieving unknown flanking DNA.
- Shows broad applicability in genomic research across different organisms.
