Question

Explain any two methods for introduction of recombinant DNA into host cell.

Answer 1

1. Electroporation: This method is based on the use of the short electrical pulses of high field strength. Electroporation causes the uptake of DNA into protoplasts by temporary permeabilization of the plasma membrane to macromolecules. Protoplasts and foreign DNA are placed in a buffer between two electrodes and a high-intensity electric current is passed, the alternating current of about 1 MHz is applied to align the protoplast by dielectrophoresis. Once aligned, fusion is induced by applying one or more direct current pulses (1-3kV /cm, 10-100 (is), then the alternating field is reapplied briefly to maintain close membrane contact for fusion (Fig. 16.1). Electric field damages membranes and creates pores in membranes. DNA diffuses through these pores immediately after the electric field is applied, until the pores are resealed. The technique is optimized by using appropriate electric field strength (defined as the applied voltage divided by the distance between two electrodes).
2. Liposomes have also been used as a carrier for the introduction of nucleic acid into plant protoplasts. Liposomes are small lipid sacs containing plasmids and are prepared artificially. The fusion of liposomes with plant protoplasts is stimulated by chemicals such as PEG (endocytosis). Liposomes mediated transformation has been achieved by including positively charged agents such as cations in the transformation mixture or using the cationic liposome preparation.