Development of an innovative and sustainable one-step method for rapid plant DNA isolation for targeted PCR using magnetic ionic liquids

Abstract

Background: Nowadays, there is an increasing demand for fast and reliable plant biomolecular analyses. Conventional methods for the isolation of nucleic acids are time-consuming and require multiple and often non-automatable steps to remove cellular interferences, with consequence that sample preparation is the major bottleneck in the bioanalytical workflow. New opportunities have been created by the use of magnetic ionic liquids (MILs) thanks to their affinity for nucleic acids.

Results: In the present study, a MIL-based magnet-assisted dispersive liquid-liquid microextraction (maDLLME) method was optimized for the extraction of genomic DNA from Arabidopsis thaliana (L.) Heynh leaves. MILs containing different metal centers were tested and the extraction method was optimized in terms of MIL volume and extraction time for purified DNA and crude lysates. The proposed approach yielded good extraction efficiency and is compatible with both quantitative analysis through fluorimetric-based detection and qualitative analysis as PCR amplification of multi and single locus genes. The protocol was successfully applied to a set of plant species and tissues.

Conclusions: The developed MIL-based maDLLME approach exhibits good enrichment of nucleic acids for extraction of template suitable for targeted PCR; it is very fast, sustainable and potentially automatable thereby representing a powerful tool for screening plants rapidly using DNA-based methods.

Keywords: Arabidopsis thaliana (L.) Heynh.; DNA barcoding; DNA isolation; Magnetic ionic liquids.

Fig. 1

Chemical structures of the investigated MILs

Fig. 2

Enrichment Factors obtained after maDLLME under different conditions with the investigated MILs for the ITS PCR products of A. thaliana

Fig. 3

Enrichment Factors obtained after maDLLME under different conditions with the investigated MILs for genomic DNA from A. thaliana previously isolated with a commercial kit

Fig. 4

Enrichment Factors obtained after maDLLME under different conditions with the investigated MILs for genomic DNA from A. thaliana plant material after simple cell lysis

Fig. 5

Amplification plots obtained by RT-PCR of the AP2D gene from A. thaliana. Blue (reference): DNA in the original sample (Ct: 24.23 min); red: DNA extracted with the maDLLLME approach and the Ni MIL (Ct: 24.11 min)

Fig. 6

PCR amplification of the nuclear ribosomal DNA (nrDNA) internal transcribed spacer (ITS) sequence (a) and the intergenic spacers of the ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (RbcL) gene (b) from A. thaliana, after extraction with the commercial kit (lane 2), the Ni-containing MIL (lanes 3 and 4), and the Co-containing MIL (lane 5 and 6). The 1 Kb DNA ladder marker is shown in lane 1. Extraction conditions: 6 µl of MIL, extraction time: 30 s

Fig. 7

Flow-chart of the proposed extraction method