Colorimetric screening of recombinant RNAi vectors without using PCR

RNAi is widely used to knock down gene expression in plants, animals and many fungi. RNAi provides a powerful technique for the derivation and analysis of loss of function of a gene. The procedure of gene knock down (silencing) includes a series of step to design a expression vector to produce hpRNA/double stranded RNA/siRNA. The vectors used for the introduction of gene fragment undergoes series of recombination and transformation. After transformation of these vector to the host bacterial cells/cell lines requires screening of desired colonies. The screening of the positive clones are generally performed with PCR. Although PCR screening is most accurate there are chances to get false positive results mostly due to cross contamination. The abstract describes about the colorimetric screening using gold nano particle (GNP). This method is robust and do not utilize PCR to screen the positive clones. In theory ssDNA (Probe) present in colloidal gold prevent the induced aggregation and confers red color. The induction is caused by addition of NaCl which screen the repulsive force among the GNP. The GNP in colloidal state is bright red whereas the fully aggregated GNP shows purple color. The separate hybridization step was conducted to scavenge complementary ssDNA probe added to the dsDNA before hybridization. The RNAi vectors containing the desired sequence binds to the probes and the solution turns blue after addition of NaCl. The Gateway Technology (Invitrogen) was used to construct the silencing vector. Briefly the target sequence for the induction of RNAi in Arabidopsis was amplified by adapter PCR and introduced into the hairpin RNA-expressing pWatergate vector (CSIRO Plant industry). A fragment of GGT2 (gamma-glutamyl-transferase) gene of 363 bp from nucleotides 104 to 467 was amplified using the primer containing attB1 and attB2 recombination sites. The amplified PCR product was first cloned into the donor vector pDONR221 (Invitrogen) to create an entry clone and after sequence verification, the gene specific sequence was transferred to the binary T-DNA destination vector pWatergate in reactions mediated by BP and LR Clonase reaction. For the screening of the recombinant plasmids, GGT2-pDONR221 and GGT2-pWATERGATE the DNA probe was designed which bind to the GGT region of the plasmid. The plasmid were isolated using the miniprep method. 50 ng of plasmid was found sufficient for the screening. This method is rapid and performed in few minutes. This method is useful in high throughput screening of the recombinant plasmid