Protocol to Identify Unknown Flanking DNA Using Partially Overlapping Primer-based PCR for Genome Walking
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...
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Bio-protocol LLC
2025-02-01
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| author | Mengya Jia Dongqin Ding Xiaohua Liu Haixing Li |
| author_facet | Mengya Jia Dongqin Ding Xiaohua Liu Haixing Li |
| author_sort | Mengya Jia |
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| description | 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. |
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| institution | Kabale University |
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| language | English |
| publishDate | 2025-02-01 |
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| spelling | doaj-art-37a8042ef88b48d18ec3a02b7592c7882025-02-07T08:16:46ZengBio-protocol LLCBio-Protocol2331-83252025-02-0115310.21769/BioProtoc.5172Protocol to Identify Unknown Flanking DNA Using Partially Overlapping Primer-based PCR for Genome WalkingMengya Jia0Dongqin Ding1Xiaohua Liu2Haixing Li3State Key Laboratory of Food Science and Resource, Nanchang University, Nanchang, ChinaSchool of Life Sciences, Lanzhou University, Lanzhou, ChinaState Key Laboratory of Food Science and Resource, Nanchang University, Nanchang, ChinaSino-German Joint Research Institute, Nanchang University, Nanchang, ChinaState Key Laboratory of Food Science and Resource, Nanchang University, Nanchang, ChinaSino-German Joint Research Institute, Nanchang University, Nanchang, ChinaState Key Laboratory of Food Science and Resource, Nanchang University, Nanchang, ChinaSino-German Joint Research Institute, Nanchang University, Nanchang, ChinaGenome 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.https://bio-protocol.org/en/bpdetail?id=5172&type=0 |
| spellingShingle | Mengya Jia Dongqin Ding Xiaohua Liu Haixing Li Protocol to Identify Unknown Flanking DNA Using Partially Overlapping Primer-based PCR for Genome Walking Bio-Protocol |
| title | Protocol to Identify Unknown Flanking DNA Using Partially Overlapping Primer-based PCR for Genome Walking |
| title_full | Protocol to Identify Unknown Flanking DNA Using Partially Overlapping Primer-based PCR for Genome Walking |
| title_fullStr | Protocol to Identify Unknown Flanking DNA Using Partially Overlapping Primer-based PCR for Genome Walking |
| title_full_unstemmed | Protocol to Identify Unknown Flanking DNA Using Partially Overlapping Primer-based PCR for Genome Walking |
| title_short | Protocol to Identify Unknown Flanking DNA Using Partially Overlapping Primer-based PCR for Genome Walking |
| title_sort | protocol to identify unknown flanking dna using partially overlapping primer based pcr for genome walking |
| url | https://bio-protocol.org/en/bpdetail?id=5172&type=0 |
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