基于融合引物的 Racket PCR 基因组步移技术的实现

Yinwei Gu; Jinfeng Pei; Mou Li; Qingchun Tang; Haixing Li

doi:10.21769/BioProtoc.5517

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In Press文章, 已正式接受发表，还没有分配卷号和期号。

Peer-reviewed

Implementation of Fusion Primer-Driven Racket PCR Protocol for Genome Walking

基于融合引物的 Racket PCR 基因组步移技术的实现

HL Haixing Li email

In Press, 发布时间: 2025年11月03日 DOI: 10.21769/BioProtoc.5517 浏览次数: 92

评审: Shengze YaoAnonymous reviewer(s)

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Abstract

Genome-walking protocols have been extensively used to clone unknown genomic sequences next to known DNAs. Existing genome-walking protocols need further improvement in methodological specificity or operation. Here, we describe a novel genome-walking protocol based on fusion primer–driven racket PCR (FPR-PCR). FPR-PCR involves four sequence-specific oligos (SSO), SSO1, SSO2, SSO3, and SSO4, which are sequentially chosen from known DNA in the direction 5’→3’. The fusion primer, mediating primary FPR-PCR, is generated by attaching SSO3 to the 5’ end of SSO1. The SSO3 encourages the target DNA of primary PCR to form a racket-like structure by mediating intra-strand annealing. SSO2 and SSO4 are directly used as sequence-specific primers (SSP) in secondary FPR-PCR, which selectively amplifies this racket-like DNA. This protocol was verified by cloning several unknown genomic sequences. Compared to traditional PCRs, FPR-PCR offers the advantages of higher specificity and fewer rounds, primarily attributed to the omission of arbitrary walking primers typically required in traditional methods.

Key features

• This FPR-PCR protocol builds upon the method constructed by Pei et al. [1].

• The FPR-PCR protocol relies on a multi-functional fusion primer (FP) that mediates the primary amplification and the formation of racket-like DNA.

• The FPR-PCR comprises only two rounds of amplification reactions.

Keywords: Genome-walking PCR (基因组步移 PCR)

Sequence-specific primer (序列特异性引物)

Fusion primer (融合引物)

Partial annealing (部分退火)

Intra-strand annealing of DNA (DNA 分子内退火)

Racket-like DNA (Racket-like DNA)

Graphical overview

Background

Genome walking (GW) is a molecular technique for identifying unknown genomic sequences next to known DNAs [2–4]. GW has been extensively used to obtain regulatory sites of genes, amplify non-conserved regions based on conserved DNAs, identify T-DNA, discover new functional genes, or screen microbes [5–7]. Therefore, GW has made significant contributions to the development of life sciences [8–10].

To date, there are many protocols for genome walking, mainly including genomic library methods, ligation-based PCRs, and arbitrary PCRs [11–14]. The genomic library method has been abandoned due to its time-consuming nature [15]. The ligation-based PCRs are also showing a tendency to be phased out, as they require pretreatment of the genomic plate prior to PCR [16–19]. In contrast, arbitrary PCRs directly mediate walking by randomly annealing a walking primer to the unknown flank; in general, the target DNA is obtained after two to three nested amplifications [20–23]. Therefore, arbitrary PCRs are a more rapid and straightforward approach. However, existing arbitrary PCRs typically realize GW by the differential amplification between target DNA and non-target DNA. Obviously, non-target background arising from walking primer challenges these PCRs [24–26].

Herein, we propose an efficient but specific genome-walking protocol, fusion primer–driven racket PCR (FPR-PCR). This method utilizes a fusion primer (FP) to mediate the target amplicon of primary FPR-PCR to form a racket-like structure; then, a secondary PCR, driven by a sequence-specific primer (SSP) pair, selectively enriches this racket-like DNA. As a result, non-target amplification is basically overcome in FPR-PCR. The FPR-PCR was validated by successfully acquiring several unknown flanking genomic DNAs [27–29].

Materials and reagents

Biological materials

1. Genome of Levilactobacillus brevis [30–35], isolated with the TIANamp Bacteria DNA kit

Reagents

1. TIANamp Bacteria DNA kit (TIANGEN, catalog number: 4992448)

2. 1× TE buffer (Sangon, catalog number: B548106)

3. LA Taq polymerase (Takara, catalog number: RR02MA)

4. 6× Loading buffer (Takara, catalog number: 9156)

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

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

7. Agarose (Sangon, catalog number: A620014)

8. 0.5 M EDTA (Solarbio, catalog number: B540625)

9. GoldView I nucleic acid staining agent (10,000 ×) (Solarbio, catalog number: G8140)

10. Tris (Solarbio, catalog number: T8060)

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

12. Oligos (Sangon)

gadC-FPα: TGTTTTCTTCTTGCTCT|ATGGTTATTCTCTGGGG

gadC-FPβ: TGTTTTCTTCTTGCTCT|TCTCTGGGGATTGATTG

gadC-SSP2: TTGGGCGTTATAATTCCTGTTTTCTTCTTG

gadC-SSP4: GGAGCGGTAGTGTGTTAGTTGGGTT

Note: The two parts (SSO1 and SSO3) in an FP are separated by a vertical line. The left part is SSO3, and the right part is SSO1.

Solutions

1. 100 μM primer (see Recipes)

2. 10 μM primer (see Recipes)

3. 2.5× TBE buffer (see Recipes)

4. 0.5× TBE buffer (see Recipes)

5. 1.5% agarose gel (see Recipes)

Recipes

1. 100 μM primer

Reagent	Final concentration	Quantity or volume
Primer powder	100 μM	n/a
1× TE buffer	1×	Volume (μL) specified by the supplier
Total	n/a	Volume (μL) specified by the supplier

2. 10 μM primer

Reagent	Final concentration	Quantity or volume
100 μM primer	10 μM	10 μL
Ultrapure water	n/a	90 μL
Total	n/a	100 μL

3. 2.5× TBE buffer (pH 8.3)

Reagent	Final concentration	Quantity or volume
Tris	225 mM	27 g
Boric acid	225 mM	13.75 g
0.5 M EDTA	5 mM	10 mL
Ultrapure water	n/a	950 mL
Total	n/a	1,000 mL

4. 0.5× TBE buffer (pH 8.3)

Reagent	Final concentration	Quantity or volume
2.5 × TBE buffer	0.5×	200 mL
Ultrapure water	n/a	800 mL
Total	n/a	1,000 mL

5. 1.5% agarose gel

Reagent	Final concentration	Quantity or volume
Agarose powder	1.5%	1.5 g
0.5× TBE buffer	0.5×	100 mL
GoldView I nucleic acid staining agent (10,000×)	1×	10 μL
Total	n/a	100 mL

Laboratory supplies

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

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

3. 0.2 mL pipette tips (Sangon, catalog number: F600227)

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

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

Equipment

1. PCR cycler (Analtytikjena, model: Biometra TOne 96G PCR)

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)

3. All data are available at https://www.frontiersin.org/articles/10.3389/fgene.2022.969840/full#supplementary-material (access date, 10/18/2022)

Procedure

English

中文翻译

文章信息

稿件历史记录

提交日期: Sep 22, 2025

接收日期: Oct 24, 2025

在线发布日期: Nov 3, 2025

版权信息

如何引用

Gu, Y., Pei, J., Li, M., Tang, Q. and Li, H. (2025). Implementation of Fusion Primer-Driven Racket PCR Protocol for Genome Walking. Bio-protocol 15(23): e5517. DOI: 10.21769/BioProtoc.5517.