PONE-D-24-43146DAPE Cloning with Modified Primers for Producing Designated Lengths of 3’ Single-Stranded Ends in PCR ProductsPLOS ONE

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Additional Editor Comments:

Reviewer 1:

In this manuscript, the authors attempt to incorporate five consecutive PT linkages to stabilize a short PCR fragment (<50bp) during T5-independent cloning. Overall, the manuscript presents some valuable insights, but several concerns must be addressed before publication:

1. Gibson assembly is a widely used method for DNA assembly, with many commercially available kits that can clone nearly any DNA fragment using T5 exonuclease, Phusion polymerase, and Taq ligase. Compared to this method, what are the main advantages of DAPE? Please elaborate on this in both the Introduction and Discussion sections. Additionally, the authors claim that inserting five consecutive PT linkages (5PT) makes the cloning end resistant to T5 exonuclease, improving the precision of the cloning process. How does this precision compare to Gibson assembly? Please design an experiment to evaluate this.

2. In Fig. 1D, the cloning efficiency significantly decreases after adding the 5PT linkages. Could the authors explain this? Also, please include a statistical analysis for this experiment. Additionally, clarify the full name of "CFU" in the figure legends.

3. Fig. 2A is identical to Fig. 1A. Please either remove it or provide additional information to differentiate it.

4. In Fig. 2B, please provide detailed conditions for the control group.

5. Fig. 3C requires statistical analysis. Please include this in the revised version.

6. On page 12, the sentence "PCR was carried out after direct primer-to-primer annealing, and then 1 μl (5%) of the PCR products was used as inserts without undergoing any DNA cleaning procedure." should be moved before "The 48 bp PCR products were reacted…".

7. Fig. 4B lacks clarity, and there is no direct comparison between the Golden Gate and DAPE groups. Please add a statistical analysis and clarify the meaning of the "Golden Gate ctl group." Ensure that all control groups are consistently labeled across all figures.

8. Fig. 5A is confusing and needs to be presented more clearly. Additionally, since EGFP is merely an example gene, it should not be given undue emphasis in the figure.

9. In Fig. 5B, 5PT actually reduces cloning efficiency in the assembly of longer fragments, as seen in Fig. 1D. Please explain this drawback of 5PT. Also, why are many of the figures missing statistical analyses?

10. Fig. 6A is unclear. Please indicate the position of the 5PT linkages in the figure

11. For comparison to lambda enzyme, if possible, please cite this reference, “Homogeneous and Sensitive Detection of microRNA with Ligase Chain Reaction and Lambda Exonuclease-Assisted Cationic Conjugated Polymer Biosensing”

Reviewer 2:

1. In the Introduction, paragraph 2, the authors state: "SLIC allows any linearizable DNA fragment to be inserted into any position of any vector of choice without incorporating additional DNA sequences, a process known as 'seamless cloning.'" However, this conclusion is limited because it does not explain the specific disadvantages of SLIC, such as the degradation of small DNA fragments due to excessive exonuclease activity. The authors should expand the discussion of the limitations of existing methods like SLIC, Gibson assembly, and Golden Gate, especially in handling small DNA fragments, and cite relevant studies or data to strengthen this point.

2. In the Introduction, paragraph 3, the authors introduce: "We introduce a versatile sequence- and ligation-independent cloning (SLIC) method called 'DNA Assembly with Phosphorothioate (PT) and T5 Exonuclease' (DAPE), which generates precise lengths of 3' overhangs at both ends of linearized DNA." This explanation is somewhat limited, as it does not elaborate on how PT-modified primers and T5 exonuclease achieve this precision. The authors should add details about how T5 exonuclease differs from other commonly used exonucleases, such as T4 DNA polymerase, and how PT-modified primers prevent excessive degradation, to help readers better understand the innovation in DAPE.

3. In the Materials and Methods section, the experimental conditions are not fully detailed, for example: "PCR samples were reacted with 1 U of diluted Lambda Exonuclease...The mixture was incubated for 90 min at 37°C before transformation." This description is too vague and might hinder reproducibility. The authors should specify all critical variables such as the exact concentration of enzymes, reaction times, and temperature controls for each step, ensuring that other researchers can replicate the experiments.

4. In the Results section, most of the experimental validation focuses on fragments smaller than 80 bp, as mentioned: "The 48 bp PCR products were reacted with 0.1 U of T5 exonuclease for 30 minutes at 30°C on a heat block." However, the effects on larger DNA fragments are not tested, which may limit the method's generalizability. The authors should include tests on larger DNA fragments (e.g., 150 bp or longer) and compare the cloning efficiency of these larger fragments with smaller ones to validate DAPE’s effectiveness across a wider range of fragment sizes.

5. In the Results section, statistical analysis is lacking, where the authors state: "Transformation efficiencies were calculated by counting the number of colonies in triplicate experiments." Although the experiments were repeated, no significance testing was performed, making it difficult to determine whether differences are meaningful. The authors should add statistical tests such as t-tests or ANOVA to evaluate the differences in cloning efficiency across conditions and indicate whether these differences are statistically significant.

6. In the Discussion section, the limitations of the DAPE method are not sufficiently addressed, for example: "Therefore, avoiding the use of DNA ligase during DNA assembly would be preferable for increasing the proportion of positive clones." While the advantages of avoiding ligase are highlighted, potential limitations are not discussed, such as the challenges DAPE may face in assembling more complex DNA constructs. The authors should add a discussion of potential limitations of the DAPE method, such as its performance in more complex genome assemblies or when handling larger DNA fragments, to provide a more balanced evaluation of the technique.

7. In the Discussion section, the comparison with other methods is not sufficiently in-depth: "DAPE, as an advanced toolkit for DNA cloning and synthetic biology, may further expedite the construction of more elaborate multi-gene circuitry." However, the actual differences between DAPE and other common methods (such as Gibson and Golden Gate) are not explored in detail. The authors should provide a more detailed comparison with other commonly used methods, particularly how DAPE performs in multi-gene assembly or with larger constructs, to better highlight its advantages in practical applications.

8. In the Figures section, the labeling and annotations are insufficient, as seen in the sentence: "Transformation efficiencies were calculated by counting the number of colonies..." The figures provide the data but lack sufficient annotations, making it difficult for readers to interpret the differences between conditions. The authors should add clearer labels and annotations for all figures, including explanations of each experimental condition and data source, to make the results more accessible to readers.

9. There are insufficient references to support the claims, for instance, when the authors mention: "Gibson-based cloning, which includes Taq ligase, caused relatively high levels of background clones." This statement is made without sufficient citation to support it. The authors should add more citations of recent studies, particularly those related to SLIC, Gibson assembly, and Golden Gate, to support the claims and improve the manuscript's credibility.

10. The experimental design lacks diversity, as only linear DNA fragments were tested: "PCR samples were reacted with 1 U of diluted Lambda Exonuclease and supplemented with 10x buffer..." No tests were conducted on other types of DNA, such as circular DNA or sequences with secondary structures. The authors should include experiments with different types of DNA (e.g., GC-rich sequences, DNA with secondary structures, or circular DNA) to test the robustness of the DAPE method in diverse scenarios.

11. The manuscript lacks discussion of practical application scenarios, for example: "DAPE, as an advanced toolkit for DNA cloning and synthetic biology, may further expedite the construction of more elaborate multi-gene circuitry." However, it does not discuss specific real-world applications, such as in CRISPR/Cas9 systems. The authors should expand the discussion to include the potential of DAPE in real-world research applications, such as CRISPR gene editing or constructing complex gene circuits, to show its practical utility and broaden its appeal.

12. The language is sometimes overly complex and the sentences are too long, as in: "T5 Exonuclease-based cloning was also known as TEDA (T5 exonuclease DNA assembly). To optimize the reaction conditions..." The long sentences may confuse readers. The authors should simplify the language and break up long sentences into shorter, more digestible parts, ensuring that each sentence conveys a single, clear point, to improve readability.

13. The results and discussion are sometimes mixed, for example: "Since the cloning efficiency of multi-piece DNA assembly by TEDA-based SLIC gradually increased with longer complementary single-stranded overhangs..." This includes both result descriptions and discussion, making the structure less clear. The authors should clearly separate the results from the discussion by placing all experimental data and observations in the "Results" section, and reserving the interpretation and analysis for the "Discussion" section, to enhance clarity.

14. The discussion of multi-fragment assembly is not detailed enough, for instance: "The relatively low yield of DAPE for assembling multiple DNA fragments could be improved by extending the 3’ overhang to over 20 bp." While the problem is mentioned, specific solutions are not discussed. The authors should expand the discussion on how to improve the efficiency of multi-fragment assembly. Consider proposing solutions such as adjusting the length of the 3’ overhangs or optimizing primer design, to provide more concrete recommendations for future improvements.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Partly

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: I Don't Know

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: In this manuscript, the authors attempt to incorporate five consecutive PT linkages to stabilize a short PCR fragment (<50bp) during T5-independent cloning. Overall, the manuscript presents some valuable insights, but several concerns must be addressed before publication:

1.Gibson assembly is a widely used method for DNA assembly, with many commercially available kits that can clone nearly any DNA fragment using T5 exonuclease, Phusion polymerase, and Taq ligase. Compared to this method, what are the main advantages of DAPE? Please elaborate on this in both the Introduction and Discussion sections. Additionally, the authors claim that inserting five consecutive PT linkages (5PT) makes the cloning end resistant to T5 exonuclease, improving the precision of the cloning process. How does this precision compare to Gibson assembly? Please design an experiment to evaluate this.

2.In Fig. 1D, the cloning efficiency significantly decreases after adding the 5PT linkages. Could the authors explain this? Also, please include a statistical analysis for this experiment. Additionally, clarify the full name of "CFU" in the figure legends.

3.Fig. 2A is identical to Fig. 1A. Please either remove it or provide additional information to differentiate it.

4.In Fig. 2B, please provide detailed conditions for the control group.

5.Fig. 3C requires statistical analysis. Please include this in the revised version.

6.On page 12, the sentence "PCR was carried out after direct primer-to-primer annealing, and then 1 μl (5%) of the PCR products was used as inserts without undergoing any DNA cleaning procedure." should be moved before "The 48 bp PCR products were reacted…".

7.Fig. 4B lacks clarity, and there is no direct comparison between the Golden Gate and DAPE groups. Please add a statistical analysis and clarify the meaning of the "Golden Gate ctl group." Ensure that all control groups are consistently labeled across all figures.

8.Fig. 5A is confusing and needs to be presented more clearly. Additionally, since EGFP is merely an example gene, it should not be given undue emphasis in the figure.

9.In Fig. 5B, 5PT actually reduces cloning efficiency in the assembly of longer fragments, as seen in Fig. 1D. Please explain this drawback of 5PT. Also, why are many of the figures missing statistical analyses?

10.Fig. 6A is unclear. Please indicate the position of the 5PT linkages in the figure

11.For comparison to lambda enzyme, if possible, please cite this reference, “Homogeneous and Sensitive Detection of microRNA with Ligase Chain Reaction and Lambda Exonuclease-Assisted Cationic Conjugated Polymer Biosensing”

Reviewer #2: 1、In the Introduction, paragraph 2, the authors state: "SLIC allows any linearizable DNA fragment to be inserted into any position of any vector of choice without incorporating additional DNA sequences, a process known as 'seamless cloning.'" However, this conclusion is limited because it does not explain the specific disadvantages of SLIC, such as the degradation of small DNA fragments due to excessive exonuclease activity. The authors should expand the discussion of the limitations of existing methods like SLIC, Gibson assembly, and Golden Gate, especially in handling small DNA fragments, and cite relevant studies or data to strengthen this point.

2、In the Introduction, paragraph 3, the authors introduce: "We introduce a versatile sequence- and ligation-independent cloning (SLIC) method called 'DNA Assembly with Phosphorothioate (PT) and T5 Exonuclease' (DAPE), which generates precise lengths of 3' overhangs at both ends of linearized DNA." This explanation is somewhat limited, as it does not elaborate on how PT-modified primers and T5 exonuclease achieve this precision. The authors should add details about how T5 exonuclease differs from other commonly used exonucleases, such as T4 DNA polymerase, and how PT-modified primers prevent excessive degradation, to help readers better understand the innovation in DAPE.

3、In the Materials and Methods section, the experimental conditions are not fully detailed, for example: "PCR samples were reacted with 1 U of diluted Lambda Exonuclease...The mixture was incubated for 90 min at 37°C before transformation." This description is too vague and might hinder reproducibility. The authors should specify all critical variables such as the exact concentration of enzymes, reaction times, and temperature controls for each step, ensuring that other researchers can replicate the experiments.

4、In the Results section, most of the experimental validation focuses on fragments smaller than 80 bp, as mentioned: "The 48 bp PCR products were reacted with 0.1 U of T5 exonuclease for 30 minutes at 30°C on a heat block." However, the effects on larger DNA fragments are not tested, which may limit the method's generalizability. The authors should include tests on larger DNA fragments (e.g., 150 bp or longer) and compare the cloning efficiency of these larger fragments with smaller ones to validate DAPE’s effectiveness across a wider range of fragment sizes.

5、In the Results section, statistical analysis is lacking, where the authors state: "Transformation efficiencies were calculated by counting the number of colonies in triplicate experiments." Although the experiments were repeated, no significance testing was performed, making it difficult to determine whether differences are meaningful. The authors should add statistical tests such as t-tests or ANOVA to evaluate the differences in cloning efficiency across conditions and indicate whether these differences are statistically significant.

6、In the Discussion section, the limitations of the DAPE method are not sufficiently addressed, for example: "Therefore, avoiding the use of DNA ligase during DNA assembly would be preferable for increasing the proportion of positive clones." While the advantages of avoiding ligase are highlighted, potential limitations are not discussed, such as the challenges DAPE may face in assembling more complex DNA constructs. The authors should add a discussion of potential limitations of the DAPE method, such as its performance in more complex genome assemblies or when handling larger DNA fragments, to provide a more balanced evaluation of the technique.

7、In the Discussion section, the comparison with other methods is not sufficiently in-depth: "DAPE, as an advanced toolkit for DNA cloning and synthetic biology, may further expedite the construction of more elaborate multi-gene circuitry." However, the actual differences between DAPE and other common methods (such as Gibson and Golden Gate) are not explored in detail. The authors should provide a more detailed comparison with other commonly used methods, particularly how DAPE performs in multi-gene assembly or with larger constructs, to better highlight its advantages in practical applications.

8、In the Figures section, the labeling and annotations are insufficient, as seen in the sentence: "Transformation efficiencies were calculated by counting the number of colonies..." The figures provide the data but lack sufficient annotations, making it difficult for readers to interpret the differences between conditions. The authors should add clearer labels and annotations for all figures, including explanations of each experimental condition and data source, to make the results more accessible to readers.

9、There are insufficient references to support the claims, for instance, when the authors mention: "Gibson-based cloning, which includes Taq ligase, caused relatively high levels of background clones." This statement is made without sufficient citation to support it. The authors should add more citations of recent studies, particularly those related to SLIC, Gibson assembly, and Golden Gate, to support the claims and improve the manuscript's credibility.

10、The experimental design lacks diversity, as only linear DNA fragments were tested: "PCR samples were reacted with 1 U of diluted Lambda Exonuclease and supplemented with 10x buffer..." No tests were conducted on other types of DNA, such as circular DNA or sequences with secondary structures. The authors should include experiments with different types of DNA (e.g., GC-rich sequences, DNA with secondary structures, or circular DNA) to test the robustness of the DAPE method in diverse scenarios.

11、The manuscript lacks discussion of practical application scenarios, for example: "DAPE, as an advanced toolkit for DNA cloning and synthetic biology, may further expedite the construction of more elaborate multi-gene circuitry." However, it does not discuss specific real-world applications, such as in CRISPR/Cas9 systems. The authors should expand the discussion to include the potential of DAPE in real-world research applications, such as CRISPR gene editing or constructing complex gene circuits, to show its practical utility and broaden its appeal.

12、The language is sometimes overly complex and the sentences are too long, as in: "T5 Exonuclease-based cloning was also known as TEDA (T5 exonuclease DNA assembly). To optimize the reaction conditions..." The long sentences may confuse readers. The authors should simplify the language and break up long sentences into shorter, more digestible parts, ensuring that each sentence conveys a single, clear point, to improve readability.

13、The results and discussion are sometimes mixed, for example: "Since the cloning efficiency of multi-piece DNA assembly by TEDA-based SLIC gradually increased with longer complementary single-stranded overhangs..." This includes both result descriptions and discussion, making the structure less clear. The authors should clearly separate the results from the discussion by placing all experimental data and observations in the "Results" section, and reserving the interpretation and analysis for the "Discussion" section, to enhance clarity.

14、The discussion of multi-fragment assembly is not detailed enough, for instance: "The relatively low yield of DAPE for assembling multiple DNA fragments could be improved by extending the 3’ overhang to over 20 bp." While the problem is mentioned, specific solutions are not discussed. The authors should expand the discussion on how to improve the efficiency of multi-fragment assembly. Consider proposing solutions such as adjusting the length of the 3’ overhangs or optimizing primer design, to provide more concrete recommendations for future improvements.

**********

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If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

**********

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DAPE Cloning with Modified Primers for Producing Designated Lengths of 3’ Single-Stranded Ends in PCR Products

PONE-D-24-43146R1

Dear Dr. Ro,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Zheng Yuan

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: I Don't Know

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: (No Response)

Reviewer #2: (No Response)

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

**********