使用PER-PCR进行基因步移挖掘未知侧翼DNA的实验方案

Zhou Yu; Dongying Wang; Zhiyu Lin; Haixing Li

doi:10.21769/BioProtoc.5188

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Protocol to Mine Unknown Flanking DNA Using PER-PCR for Genome Walking

使用PER-PCR进行基因步移挖掘未知侧翼DNA的实验方案

ZY Zhou Yu ^*

DW Dongying Wang ^*

ZL Zhiyu Lin ^*

HL Haixing Li email

(*contributed equally to this work) 发布: 2025年02月20日第15卷第4期 DOI: 10.21769/BioProtoc.5188 浏览次数: 742

评审: Alba BlesaMichael KotikAnonymous reviewer(s)

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Cover of Molecular Genetics and Genomics, featuring study using the protocol.

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Abstract

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.

Key features

• The current protocol builds upon the technique developed by Li et al. [1], which is universal to any species.

• The developed protocol relies on the partial overlap among a set of three WPs.

• Two rounds of nested PER-PCRs can generally result in a positive walking result.

Keywords: Genome walking (基因步移)

Walking primer (步移引物)

Partial overlap between walking primers (步移引物部分重叠)

Sequence-specific primer (序列特异性引物)

Partial annealing (部分退火)

Intra-strand annealing (链内退火)

Agarose gel electrophoresis (琼脂糖凝胶电泳)

DNA sequencing (DNA测序)

Graphical overview

Background

In life science–related research, genome walking has achieved various applications, such as identifying transgenes, discovering new genetic resources, and cloning full-length genes, by mining unknown genomic regions flanking known DNA [2–4]. Therefore, genome walking has played an essential role in advancing life sciences–related disciplines like genetics, genomics, and microbiology [5–8].

At present, there are many genome walking methods available. The earliest genome walking method depended on constructing and then screening a genome library, which is laborious and time-consuming. Therefore, this method is seldom adopted now [9,10]. In contrast, PCR-based genome walking has received special attention due to its rapidity, low cost, and ease of operation [11–13]. To date, dozens of PCR-based genome walking methods have been established [14–17]. The existing PCR-based methods can be divided into two groups according to whether genome preprocessing is required preceding PCR: genome preprocessing-dependent PCR (such as panhandle PCR, inverse PCR, and restriction site extension PCR) and random PCR (such as wristwatch PCR, SiteFinding-PCR, and thermal asymmetric interlaced PCR). The latter has gradually become the mainstream walking method since it is free of the time-consuming genome preprocessing step. Although there are many random PCR walking methods available now, developing a practical analogous method is still popular [1,18].

Herein, a novel protocol based on primer extension refractory PCR (PER-PCR) is detailed for rapid and reliable genome walking. The PER-PCR relies on a set of three walking primers (WP): primary WP (PWP), secondary WP (SWP), and tertiary WP (TWP). The middle 15 nt of PWP overlaps the 3' part of SWP/TWP. The 5' parts of the three WPs are different from each other. The 15 nt overlap allows the WP to anneal to its predecessor site in the low annealing temperature (50 °C) cycle. However, the WP complement is unable to be produced on the DNA template due to the mismatch of the two WPs 5' regions. Clearly, only target DNA is amplified in the next 65 °C cycles. The disadvantage of PER-PCR is the complexity of designing a practical WP set. However, the three WP sets provided in this study are universal to any species. Users need only to design the required sequence-specific primer (SSP) set. This PER-PCR protocol can be used in many aspects, such as identifying new genes, unveiling regulatory DNAs, or clarifying structures of genetic elements [1].

Materials and reagents

Biological materials

1. Genomic DNA of rice was obtained from the lab of Dr. Xiaojue Peng at Nanchang University

2. Genomic DNA of Levilactobacillus brevis CD0817 [19–22] was extracted by our lab

Reagents

1. 10× LA PCR buffer (Mg²⁺ plus) (Takara, catalog number: RR042A)

2. 6× Loading buffer (Takara, catalog number: 9156)

3. LA Taq polymerase (hot-start version) (Takara, catalog number: RR042A)

4. dNTP mixture (Takara, catalog number: RR042A)

5. DL 5,000 DNA marker (Takara, catalog number: 3428Q)

6. 1× TE buffer (Sangon, catalog number: B548106)

7. Agarose (Sangon, catalog number: A620014)

8. 1 M NaOH (Yuanye, catalog number: B28412)

9. 0.5 M EDTA (Solarbio, catalog number: B540625)

10. Green fluorescent nucleic acid dye (10,000 ×) (Solarbio, catalog number: G8140)

11. Tris (Solarbio, catalog number: T8060)

12. Boric acid (Solarbio, catalog number: B8110)

13. DiaSpin DNA Gel Extraction kit (Sangon, catalog number: B110092)

14. Primers (Sangon)

PWP1: AAAGTAGTCATGTATCTGCGTCCTAGTC

PWP2: AAAGTAGTCATGTATCTGGCAGTCATAG

PWP3: AAAGTAGTCATGTATCTGTGCTGTCTGA

SWP: GTCGGTCTGGGTAGTCATGTATCTG

TWP: ACCCTGTGCCGTAGTCATGTATCTG

SSP1-gadR: TCCTTCGTTCTTGATTCCATACCCT

SSP2-gadR: CCATTTCCATAGGTTGCTCCAAGGTCA

SSP3-gadR: TAGGATACTGGCTAAAATGAATTAACTCGGAT

SSP1-hyg: GGAAGTGCTTGACATTGGGGAGT

SSP2-hyg: AAGACCTGCCTGAAACCGAACTGC

SSP3-hyg: CAAGGAATCGGTCAATACACTACATGGC

Solutions

1. 2.5× TBE buffer (see Recipes)

2. 0.5× TBE buffer (see Recipes)

3. 100 μM primer (see Recipes)

4. 10 μM primer (see Recipes)

5. 1.5% agarose gel (see Recipes)

Recipes

1. 2.5 × TBE buffer

Reagent	Final concentration	Amount
0.5 M EDTA solution	5 mM	10 mL
Tris	225 mM	27 g
Boric acid	225 mM	13.75 g
ddH₂O	n/a	980 mL
Total	n/a	1,000 mL
Adjust pH to 8.3 with 1 M NaOH and then replenish the solution to 1,000 mL with ddH₂O.

2. 0.5× TBE buffer

Reagent	Final concentration	Amount
2.5× TBE buffer	0.5×	200 mL
ddH₂O	n/a	800 mL
Total	n/a	1,000 mL

3. 100 μM primer

Reagent	Final concentration	Quantity or Volume
Powdery primer	100 μM	n/a
1× TE buffer	1×	Volume specified in the sheet of primer synthesis
Total	n/a	Volume specified in the sheet of primer synthesis

Note: Dilute a portion of the 100 μM primer to prepare 10 μM primer and store the remaining portion at -80 °C.

4. 10 μM primer

Reagent	Final concentration	Quantity or Volume
100 μM primer	10 μM	1 μL
1× TE buffer	1×	9 μL
Total	n/a	10 μL

Note: Prepare extra volume of a 10 μM primer and divide it into multiple 1.5 mL microcentrifuge tubes; then, store the microcentrifuge tubes at -80 °C. Take one tube at a time and store it at -20 °C after use.

5. 1.5% agarose gel

Reagent	Final concentration	Quantity or Volume
Agarose	1.5%	1.5 g
0.5× TBE buffer	0.5×	100 mL
Green fluorescent nucleic acid dye (10,000×)	1×	10 μL
Total	n/a	100 mL

Laboratory supplies

1. 0.2 mL thin-wall PCR tubes (Kirgen, catalog number: KG2311)

2. 10 μL pipette tips (Sangon, catalog number: F600215)

3. 200 μL pipette tips (Sangon, catalog number: F600227)

4. 1,000 μL pipette tips (Sangon, catalog number: F630101)

5. 1.5 mL microcentrifuge tubes (Labselect, catalog number: MCT-001-150)

Equipment

1. PCR apparatus (Analtytikjena, model: Biometra TAdvanced)

2. Electrophoresis apparatus (Beijing Liuyi, model: DYY-6C)

3. Gel imaging system (Bio-Rad, model: ChemiDoc XRS+)

4. Microcentrifuge (Tiangen, model: TGear)

Software and datasets

1. Oligo 7 software (Molecular Biology Insights, Inc., USA)

2. DNASTAR Lasergene software (DNASTAR, Inc., USA)

Procedure

English

中文翻译

文章信息

稿件历史记录

提交日期: Sep 14, 2024

接收日期: Dec 12, 2024

在线发布日期: Dec 26, 2024

出版日期: Feb 20, 2025

版权信息

如何引用

Yu, Z., Wang, D., Lin, Z. and Li, H. (2025). Protocol to Mine Unknown Flanking DNA Using PER-PCR for Genome Walking. Bio-protocol 15(4): e5188. DOI: 10.21769/BioProtoc.5188.