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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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Representation of the modular assembly cloning procedure. Mutations or uncertainties are indicated as red asterisks. Sequence verified DNA is shown in green. Sequencing primers are represented by arrows.
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Figure 1: Representation of the modular assembly cloning procedure. Mutations or uncertainties are indicated as red asterisks. Sequence verified DNA is shown in green. Sequencing primers are represented by arrows.

Mentions: The traditional approach for this type of work involves amplification of the target region using a high-fidelity thermostable DNA polymerase, which usually requires optimization of the PCR conditions, but often results in a small PCR product yield owing to the low processivity of high-fidelity DNA polymerases. Moreover, in practice, the error rates can be rather high, even for proof-reading polymerases. Experimental measurement of the mutation rate in PCR products showed that for a 349-bp fragment amplified by 30 PCR cycles, approximately 1% of clones had incorrect sequences [7]. It should be noted, that a significant proportion of PCR-introduced point mutations were not detected in the study because a functional forward mutation assay was used [7]. Additionally, when higher numbers of PCR cycles are used to amplify target fragments from vertebrate genomic DNA or large target fragment sizes, this can increase the level of incorrect PCR products to tens of per cents, making identification of non-mutated clones problematic. The search for correct PCR-generated long DNA fragments is further complicated by the need for multiple specific sequencing primers directed to various regions of the target DNA sequence instead of generic vector-specific primers with known performances (Figure 1).


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)

Representation of the modular assembly cloning procedure. Mutations or uncertainties are indicated as red asterisks. Sequence verified DNA is shown in green. Sequencing primers are represented by arrows.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Representation of the modular assembly cloning procedure. Mutations or uncertainties are indicated as red asterisks. Sequence verified DNA is shown in green. Sequencing primers are represented by arrows.
Mentions: The traditional approach for this type of work involves amplification of the target region using a high-fidelity thermostable DNA polymerase, which usually requires optimization of the PCR conditions, but often results in a small PCR product yield owing to the low processivity of high-fidelity DNA polymerases. Moreover, in practice, the error rates can be rather high, even for proof-reading polymerases. Experimental measurement of the mutation rate in PCR products showed that for a 349-bp fragment amplified by 30 PCR cycles, approximately 1% of clones had incorrect sequences [7]. It should be noted, that a significant proportion of PCR-introduced point mutations were not detected in the study because a functional forward mutation assay was used [7]. Additionally, when higher numbers of PCR cycles are used to amplify target fragments from vertebrate genomic DNA or large target fragment sizes, this can increase the level of incorrect PCR products to tens of per cents, making identification of non-mutated clones problematic. The search for correct PCR-generated long DNA fragments is further complicated by the need for multiple specific sequencing primers directed to various regions of the target DNA sequence instead of generic vector-specific primers with known performances (Figure 1).

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