KS
Keyi Shen
  • Ph. D. Candidate, Department of Structural and Cellular Biology, Tulane University, New Orleans, USA
Research fields
  • Biochemistry, Cancer Biology, Cell Biology, Molecular Biology
Protocol to Mine Unknown Flanking DNA Using PER-PCR for Genome Walking
Authors:  Zhou Yu, Dongying Wang, Zhiyu Lin and Haixing Li, date: 02/20/2025, view: 51, Q&A: 0

Genome walking, a molecular technique for mining unknown flanking DNAs, has a wide range of uses in life sciences and related areas. Herein, a simple but reliable genome walking protocol named primer extension refractory PCR (PER-PCR) is detailed. This PER-PCR-based protocol uses a set of three walking primers (WPs): primary WP (PWP), secondary WP (SWP), and tertiary WP (TWP). The 15 nt middle region of PWP overlaps the 3' region of SWP/TWP. The 5' regions of the three WPs are completely different from each other. In the low annealing temperature cycle of secondary or tertiary PER-PCR, the short overlap mediates the annealing of the WP to the previous WP site, thus producing a series of single-stranded DNAs (ssDNA). However, the 5' mismatch between the two WPs prevents the template DNA from synthesizing the WP complement at its 3' end. In the next high annealing temperature cycles, the target ssDNA is exponentially amplified because it is defined by both the WP and sequence-specific primer, while non-target ssDNA cannot be amplified as it lacks a binding site for at least one of the primers. Finally, the target DNA becomes the main PER-PCR product. This protocol has been validated by walking two selected genes.

Protocol to Identify Unknown Flanking DNA Using Partially Overlapping Primer-based PCR for Genome Walking
Authors:  Mengya Jia, Dongqin Ding, Xiaohua Liu and Haixing Li, date: 02/05/2025, view: 106, Q&A: 0

Genome walking is a popular molecular technique for accessing unknown flanking DNAs, which has been widely used in biology-related fields. Herein, a simple but accurate genome-walking protocol named partially overlapping primer (POP)-based PCR (POP-PCR) is described. This protocol exploits a POP set of three POPs to mediate genome walking. The three POPs have a 10 nt 3' overlap and 15 nt heterologous 5' regions. Therefore, a POP can partially anneal to the previous POP site only at a relatively low temperature (approximately 50 °C). In primary POP-PCR, the low-temperature (25 °C) cycle allows the primary POP to partially anneal to site(s) of an unknown flank and many sites of the genome, synthesizing many single-stranded DNAs. In the subsequent high-temperature (65 °C) cycle, the target single-stranded DNA is converted into double-stranded DNA by the sequence-specific primer, attributed to the presence of this primer complement, while non-target single-stranded DNA cannot become double-stranded because it lacks a binding site for both primers. As a result, only the target DNA is amplified in the remaining 65 °C cycles. In secondary or tertiary POP-PCR, the 50 °C cycle directs the POP to the previous POP site and synthesizes many single-stranded DNAs. However, as in the primary PCR, only the target DNA can be amplified in the subsequent 65 °C cycles. This POP-PCR protocol has many potential applications, such as screening microbes, identifying transgenic sites, or mining new genetic resources.

Protocol to Retrieve Unknown Flanking DNA Using Fork PCR for Genome Walking
Authors:  Hongjing Wu, Hao Pan and Haixing Li, date: 01/20/2025, view: 1185, Q&A: 0

PCR-based genome walking is one of the prevalent techniques implemented to acquire unknown flanking genomic DNAs. The worth of genome walking includes but is not limited to cloning full-length genes, mining new genes, and discovering regulatory regions of genes. Therefore, this technique has advanced molecular biology and related fields. However, the PCR amplification specificity of this technique needs to be further improved. Here, a practical protocol based on fork PCR is proposed for genome walking. This PCR uses a fork primer set of three arbitrary primers to execute walking amplification task, where the primary fork primer mediates walking by partially annealing to an unknown flank, and the fork-like structure formed between the three primers participates in inhibiting non-target amplification. In primary fork PCR, the low-annealing temperature (25 °C) cycle allows the primary fork primer to anneal to many sites of the genome, synthesizing a cluster of single-stranded DNAs; the subsequent 65 °C cycle processes the target single-strand into double-strand via the site-specific primer; then, the remaining 65 °C cycles selectively enrich this target DNA. However, any non-target single-stranded DNA formed in the 25 °C cycle cannot be further processed in the following 65 °C cycles because it lacks an exact binding site for any primer. Secondary, or even tertiary nested fork PCR further selectively enriches the target DNA. The practicability of fork PCR was validated by walking three genes in Levilactobacillus brevis CD0817 and one gene in Oryza sativa. The results indicated that the proposed protocol can serve as a supplement to the existing genome walking protocols.

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