Bridging PCR-Based Genome-Walking Protocol

Materials and reagents

Biological materials

1. Genome of Levilactobacillus brevis CD0817 [28–33], extracted using the Bacterial Genomic DNA Isolation kit (Tiangen Biotech Co., Ltd., Beijing, China)

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. Goldview nucleic acid gel stain (10,000×) (Yeasen, catalog number: 10201ES03)

11. Tris (Solarbio, catalog number: T8060)

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

13. TaKaRa MiniBEST DNA Fragment Purification kit v4.0 (TaKaRa, catalog number: DV9761)

14. Primers (Sangon)

WP1: GTCGTAGTCATGTATCGTCCTAGTCATCTGCTTGTTCGTCAGTCAGCGTC

WP2: GTCGTAGTCATGTATCGTCCTAGTCTCAGTCAGTCAGTTGCAGTCAGTCT

WP3: GTCGTAGTCATGTATCGTCCTAGTCATCCAGAACAGTCGATTGGTTCAGC

BP: CAGTCAGTCTCAGCTAGTCAGTGTCGTCGTAGTCATGTATCGTCCTAGTC

TP: CAGTCAGTCTCAGCTAGTCAGTGTC

gadA-SSP1: TCCAAGAATCATCCGCAATCGTCA

gadA-SSP2: TGGTAACATCGTCACGGTTCTTTGG

gadA-SSP3: TAGCCTTGTACCCATCTTTACCGAA

gadR-SSP1: TCCTTCGTTCTTGATTCCATACCCT

gadR-SSP2: CCATTTCCATAGGTTGCTCCAAGG

gadR-SSP3: GGATACTGGCTAAAATGAATTAACTCGGATAA

hyg-SSP1: ACGGCAATTTCGATGATGCAGCTTG

hyg-SSP2: GGGACTGTCGGGCGTACACAA

hyg-SSP3: CTGGACCGATGGCTGTGTAGAAG

Solutions

1. 2.5× TBE buffer (see Recipes)

2. 0.5× TBE buffer (see Recipes)

3. 100 mM primer (see Recipes)

4. 10 mM primer (see Recipes)

5. 1.5% agarose gel (see Recipes)

Recipes

1. 2.5× TBE buffer (pH 8.3)

This 2.5× TBE buffer can be stored at room temperature for 3 months.

2. 0.5× TBE buffer (pH 8.3)

This 0.5× TBE buffer can be stored at room temperature for 3 months.

3. 100 μM primer

Note: Take 10 μL of this primer solution to make 10 μM primer, and store the remaining solution at -80 °C.

4. 10 μM primer

Note: Divide 10 μM primer into 10 μL/tube, then store these tubes at -20 °C.

5. 1.5% agarose gel

Laboratory supplies

1. 0.2 mL 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. 1,500 μL microcentrifuge tubes (Labselect, catalog number: MCT-001-150)

Equipment

1. PCR apparatus (Applied Biosystems, model: Biometra TAdvanced 96 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 v7.37 software (Molecular Biology Insights, Inc., USA)

2. DNASTAR Lasergene v7.1 software (DNASTAR, Inc., USA)

Procedure

A. Design of primers

The three WP sets used in this study are presented in Figure 1.

Figure 1. Three walker primer (WP) sets used in this study and the interrelationship of WP, bridging primer (BP), and tail primer (TP) in a WP set.

Critical: WP (50 nt), BP (50 nt), and TP (25 nt) are random oligo DNAs, with Tm values of 70–75, 70–75, and 60–65 °C, respectively. BP is made by attaching TP to the 5′ end of WP 5′ region.

Note: Design three WP sets so as to perform three parallel sets of bridging PCRs in a WP. The three WPs have an identical 5′ region (25 nt) but completely different 3′ regions (25 nt). The identical 5′ region means that only one BP and one TP are required, while the different 3′ regions endow WPs with individualized annealing patterns. Therefore, the three WP sets are actually constituted by five primers, namely, three WPs, one BP, and one TP.

1. Design of WP.

a. Open the Oligo 7 software, click File and New Sequence to show Edit Sequence dialog box; type in a 50 nt arbitrary sequence in Edit Sequence dialog box (Figure 2A) and then sequentially click Accept/Discard and Accept to accept the input sequence (Figure 2B).

Figure 2. Screenshots showing how to input an arbitrary primer sequence. (A) Discovery of Edit Sequence dialog box. (B) How to accept the input sequence.

b. Click Analyze, Duplex Formation, and Current Oligo (Figure 3A) to assess primer dimer(s) (Figure 3B).

Figure 3. Screenshots showing how to assess primer dimer(s). (A) Discovery of Duplex Formation and Current Oligo under Analyze tab. (B) Output dimer(s).

c. Click Analyze, Hairpin Formation, and Current Oligo (Figure 4A) to assess primer hairpin(s) (Figure 4B).

Figure 4. Screenshots showing how to check primer hairpin(s). (A) Discovery of Hairpin Formation and Current Oligo under Analyze tab. (B) Output hairpin(s).

Note: Edit this primer and then re-assess it if it shows a severe dimer(s) or hairpin(s) with Tm ≥ 40 °C.

d. Return to the Edit Sequence dialog box (Figure 2A) by clicking Edit and Entire Sequence (Figure 5A). Change the sequence according to the above analysis outcomes; click Accept/Discard and Accept (Figure 2B) and then minimize this dialog box to show the dialog box shown in Figure 3A.

Figure 5. Screenshots showing how to optimize the primer. (A) Discovery of Edit Sequence dialog box. (B) Output primer hairpin(s).

e. Repeat steps A1b–c to evaluate the sequence until satisfactory WP (Figure 5B) is obtained.

Notes:

1. The WP shown in Figure 5B is acceptable because it has no obvious primer dimer or hairpin.

2. Fix the 5′ region (25 nt) of this WP, then add another arbitrary sequence (25 nt) to its 3′ end; repeat steps A1b–c to evaluate this new primer until it is satisfactory. Three WPs are designed in this study.

2. Design a satisfactory TP by repeating steps A1a–e, just like designing the WP.

3. Create a BP by attaching the TP to the 5′ end of WP 5′ region (25 nt).

Note: Assess this BP by repeating steps A1b–c until a satisfactory BP is created.

4. Pick SSP.

a. Open the Oligo 7 software and click File and Open to input the known DNA sequence.

b. Click Change and Current Oligo Length to select and define the length of SSP (Figure 6A).

c. Assess the SSP by repeating steps A1b–c (Figure 6B).

Note: Redesign the SSP and evaluate it by repeating steps A1b–c until a satisfactory one (Figure 6B) is obtained, if the current SSP forms a severe primer dimer(s) or hairpin(s) with Tm ≥ 40 °C.

Figure 6. Screenshots showing how to pick a sequence-specific primer (SSP). (A) Discovery of Current Oligo Length. (B) Output primer dimer(s) and hairpin(s).

Critical: The SSP should have a Tm of 60–65 °C according to Mazars et al. [34].

B. Bridging PCR procedure

A bridging PCR set contains three rounds of nested PCR. The primary PCR is driven by WP and SSP1, the secondary PCR is driven by TP, BP, and SSP2, and the tertiary PCR is driven by TP and SSP3 (Figure 7).

Figure 7. Schematic diagram of bridging PCR. WP: walker primer; BP: bridging primer; TP: tail primer; SSP: sequence-specific primer. Thin solid lines: known sequences; thin dotted lines: unknown sequences.

Critical: The working concentration of BP is only one twenty-fifth of TP or SSP2.

1. Primary bridging PCR.

a. Pipette primary bridging PCR components (Table 1) into a PCR tube.

Table 1. Primary bridging PCR mix

b. Mix the components well.

c. Centrifuge the tube at 3,000× g for 20 s at 4 °C.

d. Run PCR amplification (Table 2).

Table 2. Primary bridging PCR cycling conditions

e. Take 1 μL of the product as template for secondary bridging PCR.

f. Store the remaining product at -20 °C.

2. Secondary bridging PCR.

a. Pipette secondary bridging PCR components (Table 3) into a PCR tube.

Table 3. Secondary bridging PCR mix

Critical: The working concentration of BP is only one twenty-fifth of TP or SSP2.

Note: If necessary, dilute the primary bridging PCR product 10–1,000 times.

b. Mix the components well.

c. Centrifuge the tube at 3,000× g for 20 s at 4 °C.

d. Run PCR amplification (Table 4).

Table 4. Secondary bridging PCR cycling conditions

e. Put the PCR product onto ice.

f. Take 1 μL of the product as the template for tertiary bridging PCR.

g. Store the remaining product at -20 °C.

3. Tertiary bridging PCR.

a. Pipette tertiary bridging PCR components (Table 5) into a PCR tube.

Table 5. Tertiary bridging PCR mix

Critical: Dilute the secondary bridging PCR product 10–1,000 times if necessary.

b. Mix the components well.

c. Centrifuge at 3,000× g for 20 s at 4 °C.

d. Run PCR amplification (Table 6).

Table 6. Tertiary fork PCR cycling conditions

e. Store the PCR product at -20 °C.

C. Electrophoresis

1. Add 5 μL of each bridging PCR product and 1 μL of 6× loading buffer.

2. Transfer the mixture into a 1.5% agarose gel supplemented with 1× Goldview nucleic acid gel stain.

3. Electrophorese at a voltage of 5 V/cm for 30 min.

4. Check the gel using the ChemiDoc XRS+ imaging system (Figure 8).

Figure 8. Genome walking of gadA (A) and gadR (B) of Levilactobacillus brevis CD 0817 and hyg (C) of rice. WP1, WP2, and WP3 denote the three parallel bridging PCR sets. P: primary PCR; S: secondary PCR; T: Tertiary PCR; and M: DNA5000 Marker. The white arrowheads indicate the target DNA bands.

D. Recovery of PCR product

1. Mix 40 μL of secondary/tertiary bridging PCR product and 8 μL of 6× loading buffer.

2. Transfer the mixture into a 1.5% agarose gel supplemented with 1× Goldview nucleic acid gel stain.

3. Electrophorese at a voltage of 5 V/cm for 30 min.

4. Check the gel using the ChemiDoc XRS+ imaging system (Figure 9) and cut out the target DNA band(s) with a knife.

5. Purify the DNA band(s) from the cut gel using the Mini-BEST Agarose Gel DNA Extraction kit v4.0.

E. DNA sequencing

Mail the purified product(s) to Sangon Biotech Co., Ltd for sequencing.

Data analysis

1. Analyze the sequencing data using the MegAlign software.

a. Open the software, then click File and Enter Sequences to input DNA sequences to be analyzed (Figure 9).

Figure 9. Screenshots showing how to input DNA sequences

b. Click Align and By Clustal W Method (Figure 10A) to get the result (Figure 10B).

Note: The experiment is considered successful if the SSP3-sided segment of the bridging PCR product overlaps the known DNA (Figure 10B).

Figure 10. Screenshots showing how to analyze the input sequences. (A) Discovery of By Clustal W Method. (B) Final outcome.

Validation of protocol

This protocol or parts of it has been used and validated in the following research articles:

Lin et al. [1]. Bridging PCR: An Efficient and Reliable Scheme Implemented for genome walking. Current Issues in Molecular Biology (Figure 8).

General notes and troubleshooting

General notes

1. The bridging PCR protocol is a universal genome-walking tool.

2. Secondary bridging PCR amplification can generally release a positive result.

3. Simultaneously performing parallel bridging PCRs will improve the success and efficiency of genome walking.

4. In secondary bridging PCR, the working concentration of BP is very low, and its role is just to introduce a TP sequence to the 5′ end of the WP 5′ region. The real amplifiers are TP and SSP3.

5. The ends of non-target DNA primed by WP are lengthened by BP. This DNA cannot be amplified by TP in the next PCR, because it forms a hairpin structure via the lengthened ends.

Troubleshooting

Problem 1: Secondary/tertiary bridging PCR does not produce target DNA(s).

Possible causes: Non-target amplification efficiency is high, or target amplification is insufficient.

Solutions: Dilute the previous product properly and use it as the template for the next PCR. If this is still ineffective, redesign an SSP set.

Problem 2: DNA band(s) cannot be directly sequenced.

Possible cause: There may be interference from non-target background.

Solution: T-clone the target DNA band and then sequence [35].

Problem 3: The DNA band(s) are not the target.

Possible cause: There may be sites homologous to SSP(s) in other regions of the genome.

Solution: Redesign an SSP set.

Acknowledgments

This study was supported by the State Key Laboratory of Food Science and Resources (SKLF-ZZB-202523), Nanchang University, China, and the Jiangxi Provincial Department of Science and Technology (20225BCJ22023), China. This protocol has been originally described and validated in Current Issues in Molecular Biology [1].

Competing interests

The authors declare no competing interests.

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