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

BMC Research Notes

Ivan I Vorobiev²  Alexandre V Orlov¹  Nadezhda A Orlova² 

[1] “Datanium” Jsc, Lazorevyj proezd, 3 Bld. 5, Moscow 129323, Russia;Laboratory of Biocatalysis, Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya 16/10, Moscow 119971, Russia

关键词: Hybrid restriction site;    Cloning technique;    Software tool;    Molecular cloning;   

Others  :  1166285
DOI  :  10.1186/1756-0500-5-303

 received in 2012-03-27, accepted in 2012-06-08,  发布年份 2012

【 摘 要 】

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.

【 授权许可】

   
2012 Orlova et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

Figure 2.

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