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A modular assembly cloning technique (aided by the BIOF software tool) for seamless and error-free assembly of long DNA fragments.

Orlova NA, Orlov AV, Vorobiev II - BMC Res Notes (2012)

Bottom Line: MAC was tested on a practical dataset, namely, two non-coding fragments of the translation elongation factor 1 alpha gene from Chinese hamster ovary cells.The individual fragment lengths exceeded 5 kbp, and direct PCR amplification produced no amplicons.This approach may be used to generate long artificial DNA fragments such as in vitro spliced cDNAs.

View Article: PubMed Central - HTML - PubMed

Affiliation: Russian Academy of Sciences, Moscow, Russia. nobiol@gmail.com

ABSTRACT

Background: Molecular cloning of DNA fragments >5 kbp is still a complex task. When no genomic DNA library is available for the species of interest, and direct PCR amplification of the desired DNA fragment is unsuccessful or results in an incorrect sequence, molecular cloning of a PCR-amplified region of the target sequence and assembly of the cloned parts by restriction and ligation is an option. Assembled components of such DNA fragments can be connected together by ligating the compatible overhangs produced by different restriction endonucleases. However, designing the corresponding cloning scheme can be a complex task that requires a software tool to generate a list of potential connection sites.

Findings: The BIOF program presented here analyzes DNA fragments for all available restriction enzymes and provides a list of potential sites for ligation of DNA fragments with compatible overhangs. The cloning scheme, which is called modular assembly cloning (MAC), is aided by the BIOF program. MAC was tested on a practical dataset, namely, two non-coding fragments of the translation elongation factor 1 alpha gene from Chinese hamster ovary cells. The individual fragment lengths exceeded 5 kbp, and direct PCR amplification produced no amplicons. However, separation of the target fragments into smaller regions, with downstream assembly of the cloned modules, resulted in both target DNA fragments being obtained with few subsequent steps.

Conclusions: Implementation of the MAC software tool and the experimental approach adopted here has great potential for simplifying the molecular cloning of long DNA fragments. This approach may be used to generate long artificial DNA fragments such as in vitro spliced cDNAs.

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Related in: MedlinePlus

BIOF program workflow. In the upper pane (A) the DNA sequence being analyzed is displayed. In the lower pane (B) the list of imported restriction endonucleases is displayed; this can be switched to the list of hybrid sites found in the DNA sequence.
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Figure 2: BIOF program workflow. In the upper pane (A) the DNA sequence being analyzed is displayed. In the lower pane (B) the list of imported restriction endonucleases is displayed; this can be switched to the list of hybrid sites found in the DNA sequence.

Mentions: The BIOF application workflow begins by loading the FASTA-formatted or plain text file with the target DNA sequence (Figure 2). Alternatively, the DNA sequence may be pasted from the clipboard into the upper pane of the application; multiple copy–paste operations and editing of the DNA sequence may be performed within the pane. The application adheres to the FASTA format description [9] and removes non-relevant symbols such as numbers and spaces from the sequence data automatically. Mapping palindromes and hybrid restriction sites starts when the user presses the button “Execute!”. The positions of the sites found by the software are displayed in the lower pane; palindromic restriction sites (i.e. sites that are recognized by one RE) are marked by asterisks. Lists of the sites found are automatically saved in the text file for further use.


A modular assembly cloning technique (aided by the BIOF software tool) for seamless and error-free assembly of long DNA fragments.

Orlova NA, Orlov AV, Vorobiev II - BMC Res Notes (2012)

BIOF program workflow. In the upper pane (A) the DNA sequence being analyzed is displayed. In the lower pane (B) the list of imported restriction endonucleases is displayed; this can be switched to the list of hybrid sites found in the DNA sequence.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3539906&req=5

Figure 2: BIOF program workflow. In the upper pane (A) the DNA sequence being analyzed is displayed. In the lower pane (B) the list of imported restriction endonucleases is displayed; this can be switched to the list of hybrid sites found in the DNA sequence.
Mentions: The BIOF application workflow begins by loading the FASTA-formatted or plain text file with the target DNA sequence (Figure 2). Alternatively, the DNA sequence may be pasted from the clipboard into the upper pane of the application; multiple copy–paste operations and editing of the DNA sequence may be performed within the pane. The application adheres to the FASTA format description [9] and removes non-relevant symbols such as numbers and spaces from the sequence data automatically. Mapping palindromes and hybrid restriction sites starts when the user presses the button “Execute!”. The positions of the sites found by the software are displayed in the lower pane; palindromic restriction sites (i.e. sites that are recognized by one RE) are marked by asterisks. Lists of the sites found are automatically saved in the text file for further use.

Bottom Line: MAC was tested on a practical dataset, namely, two non-coding fragments of the translation elongation factor 1 alpha gene from Chinese hamster ovary cells.The individual fragment lengths exceeded 5 kbp, and direct PCR amplification produced no amplicons.This approach may be used to generate long artificial DNA fragments such as in vitro spliced cDNAs.

View Article: PubMed Central - HTML - PubMed

Affiliation: Russian Academy of Sciences, Moscow, Russia. nobiol@gmail.com

ABSTRACT

Background: Molecular cloning of DNA fragments >5 kbp is still a complex task. When no genomic DNA library is available for the species of interest, and direct PCR amplification of the desired DNA fragment is unsuccessful or results in an incorrect sequence, molecular cloning of a PCR-amplified region of the target sequence and assembly of the cloned parts by restriction and ligation is an option. Assembled components of such DNA fragments can be connected together by ligating the compatible overhangs produced by different restriction endonucleases. However, designing the corresponding cloning scheme can be a complex task that requires a software tool to generate a list of potential connection sites.

Findings: The BIOF program presented here analyzes DNA fragments for all available restriction enzymes and provides a list of potential sites for ligation of DNA fragments with compatible overhangs. The cloning scheme, which is called modular assembly cloning (MAC), is aided by the BIOF program. MAC was tested on a practical dataset, namely, two non-coding fragments of the translation elongation factor 1 alpha gene from Chinese hamster ovary cells. The individual fragment lengths exceeded 5 kbp, and direct PCR amplification produced no amplicons. However, separation of the target fragments into smaller regions, with downstream assembly of the cloned modules, resulted in both target DNA fragments being obtained with few subsequent steps.

Conclusions: Implementation of the MAC software tool and the experimental approach adopted here has great potential for simplifying the molecular cloning of long DNA fragments. This approach may be used to generate long artificial DNA fragments such as in vitro spliced cDNAs.

Show MeSH
Related in: MedlinePlus